Compositions and methods for transgene expression

Abstract

Described herein are compositions for modulating transgene expression. Also described herein are methods for using the compositions described herein to modulate transgene expression.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 236,168, filed August 23, 2021, the entirety of which is hereby incorporated by reference into this specification.

[0002] Incorporation by Reference

[0002] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. To the extent that the publications and patents or patent applications incorporated herein by reference conflict with the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such conflicting material. [Background technology]

[0003]

[0003] Neovascularization, including vasculogenesis, angiogenesis and arteriogenesis, is regulated by a variety of cell signaling pathways. One of the signaling pathways is regulated by vascular endothelial growth factor (VEGF). There are four major types of VEGF, including VEGF-A, VEGF-B, VEGF-C and VEGF-D. There are many isoforms of VEGF-A resulting from alternative splicing of mRNA derived from VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189 and VEGF206. VEGF is a potent mitogen for endothelial cells, inducing proliferation, migration, vascular tube formation and permeability. Thus, increasing the VEGF signaling pathway increases neovascularization signals, while decreasing or inhibiting the VEGF signaling pathway decreases neovascularization signals.

[0004]

[0004] VEGF inhibition is one of the most common treatment options for diseases or conditions related to neovascularization. Current treatment with VEGF inhibitors can be cumbersome due to the short half-life of VEGF inhibitors, which requires repeated monthly injections to achieve and sustain the suppression of neovascularization. Therefore, it has become increasingly clear that the full potential of VEGF inhibition can only be realized by increasing the therapeutic effect of VEGF inhibition. Summary of the Invention [Means for solving the problem]

[0005]

[0005] There remains a need for biological agents that modulate signaling transduction in ligand and receptor interactions associated with neovascularization, thus complementing or producing a synergistic therapeutic effect when combined with VEGF inhibition. Thus, described herein are non-naturally occurring polynucleotides that include one or more expression cassettes encoding VEGF inhibitors and signaling transduction regulators (e.g., activators of receptor tyrosine kinases associated with VEGF signaling) that enhance and complement the therapeutic effect of VEGF inhibition. Such combinations can synergistically increase the therapeutic effect of VEGF inhibition and reduce neovascularization signaling.

[0006]

[0006] In some aspects, described herein are non-naturally occurring polynucleotides comprising one or more expression cassettes for expressing a VEGF inhibitor; and a receptor tyrosine kinase (RTK) / Tie2 or an activator of RTK / Tie2. In some embodiments, the VEGF inhibitor and the RTK / Tie or activator of RTK / Tie2 are expressed as separate polypeptides, or as contiguous polypeptides cleavable into separate polypeptides, comprising the VEGF inhibitor and the RTK / Tie2 or activator of RTK / Tie2. In some embodiments, the contiguous polypeptide comprises a protease-cleavable sequence. In some embodiments, the contiguous polypeptide comprises a furin-cleavable sequence. In some embodiments, the contiguous polypeptide comprises an auto-cleaving polypeptide sequence. In some embodiments, the auto-cleaving polypeptide sequence comprises a 2A auto-cleaving peptide. In some embodiments, the auto-cleaving polypeptide sequence comprises an F2A auto-cleaving peptide. In some embodiments, the protease-cleavable sequence comprises a furin-F2A cleavage sequence. In some embodiments, the VEGF inhibitor binds to and inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF inhibitor comprises an antibody. In some embodiments, the VEGF inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the VEGF inhibitor comprises a non-antibody VEGF inhibitor. In some embodiments, the non-antibody VEGF inhibitor is a VEGF receptor 1 (VEGFR1), a VEGF receptor 2 (VEGFR2), a VEGF receptor 3 (VEGFR3), a fragment thereof, or a combination thereof.In some embodiments, the non-antibody VEGF inhibitor comprises soluble VEGFR1, soluble VEGFR2, soluble VEGFR3, soluble fragments thereof, or combinations thereof. In some embodiments, the non-antibody VEGF inhibitor comprises VEGF-Trap or a modified version thereof. In some embodiments, the activator of RTK / Tie2 comprises angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), angiopoietin-3 (Ang-3), or angiopoietin-4 (Ang-4). In some embodiments, the activator of RTK / Tie2 comprises Ang1. In some embodiments, Ang1 comprises full-length Ang1. In some embodiments, Ang1 comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3. In some embodiments, Ang1 comprises a functional fragment of Ang1. In some embodiments, the functional fragment of Ang1 comprises a fibronectin-like domain (FLD). In some embodiments, the FLD comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:5. In some embodiments, the FLD is fused to a soluble polypeptide. In some embodiments, the soluble polypeptide comprises a polypeptide sequence that is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, or at most 93% identical to SEQ ID NO:1. In some embodiments, the soluble polypeptide comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:2. In some embodiments, the soluble polypeptide comprises a polypeptide sequence that is SEQ ID NO:2. In some embodiments, the activator of RTK / Tie2 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 6. In some embodiments, the activator of RTK / Tie2 comprises an antibody or a fragment thereof.In some embodiments, the activator of RTK / Tie2 comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the activator of RTK / Tie2 binds to and inhibits Ang2. In some embodiments, the antibody or fragment thereof binds to a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the antibody or fragment thereof comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 25-27, a fragment thereof, or a combination thereof. In some embodiments, the activator of RTK / Tie2 comprises an inhibitory RNA. In some embodiments, the inhibitory RNA comprises an shRNA, an siRNA, an miRNA, or a combination thereof. In some embodiments, the inhibitory RNA comprises an shRNA. In some embodiments, the inhibitory RNA binds to an endogenous nucleic acid encoding an angiopoietin. In some embodiments, the angiopoietin comprises Ang2. In some embodiments, the Ang2 comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the one or more expression cassettes comprise one or more promoters, one or more internal ribosome entry sites (IRES), or both.In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce neoangiogenic signaling by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more when expressed in a cell compared to neoangiogenic signaling in the absence of the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the neoangiogenic signaling reduced by a comparable VEGF inhibitor and a comparable RTK / Tie2 activator or a comparable RTK / Tie2 encoded from two different non-naturally occurring polynucleotides.

[0007]

[0007] In some aspects, described herein is a non-naturally occurring polynucleotide comprising one or more expression cassettes for expressing a VEGF inhibitor; and an Ang1 polypeptide. In certain aspects, also described herein is a non-naturally occurring polynucleotide comprising one or more expression cassettes for expressing a VEGF inhibitor; and an Ang2 inhibitor. In some embodiments, the VEGF inhibitor binds to and inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF inhibitor comprises an antibody. In some embodiments, the VEGF inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the VEGF inhibitor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or a combination or fragment thereof. In some embodiments, the VEGF inhibitor comprises a non-antibody VEGF inhibitor. In some embodiments, the non-antibody VEGF inhibitor comprises VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, a fragment thereof or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises a soluble VEGFR1, a soluble VEGFR2, a soluble VEGFR3, a soluble fragment thereof or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises VEGF-Trap or a modified version thereof.In some embodiments, the non-antibody VEGF inhibitor comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:31, or a combination thereof or a fragment thereof. In some embodiments, the Ang1 polypeptide is full-length Ang1. In some embodiments, the Ang1 polypeptide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:3. In some embodiments, the Ang1 polypeptide comprises an Ang1 functional fragment that includes the fibronectin-like domain (FLD) of Ang1. In some embodiments, the Ang1 polypeptide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:5. In some embodiments, the FLD is fused to a soluble polypeptide comprising a polypeptide sequence that is at most 99%, at most 98%, at most 96%, at most 95%, at most 94% or at most 93% identical to SEQ ID NO:6. In some embodiments, the Ang2 inhibitor comprises an antibody or a fragment thereof that binds and inhibits Ang2. In some embodiments, the Ang2 inhibitor comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or combinations thereof, binding fragments thereof, or chemically modified derivatives thereof. In some embodiments, the antibody or a fragment thereof binds to a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:12.In some embodiments, the antibody or fragment comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 25-27, or a fragment thereof, or a combination thereof. In some embodiments, the Ang2 inhibitor comprises RNA interference (RNAi). In some embodiments, the RNAi comprises shRNA, siRNA, miRNA, or a combination thereof. In some embodiments, the RNAi comprises an shRNA that binds to an endogenous nucleic acid encoding Ang2. In some embodiments, the RNAi binds to an Ang2 nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the non-naturally occurring polynucleotide comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NOs: 81-86. In some embodiments, the non-naturally occurring polynucleotide comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NOs: 31-34 and 51-77. In some embodiments, the one or more expression cassettes comprise one or more promoters, one or more internal ribosome entry sites (IRES), or both. In some embodiments, the VEGF inhibitor and the Ang1 polypeptide reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neoangiogenic signaling in the absence of the VEGF inhibitor and the Ang1 polypeptide.In some embodiments, the VEGF inhibitor and the Ang1 polypeptide reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the neoangiogenic signaling reduced by a comparable VEGF inhibitor and a comparable Ang1 polypeptide encoded from two different non-naturally occurring polynucleotides. In some embodiments, the VEGF inhibitor and the Ang2 inhibitor reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the neoangiogenic signaling in the absence of the VEGF inhibitor and the Ang2 inhibitor. In some embodiments, the VEGF inhibitor and the Ang2 inhibitor reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the neoangiogenic signaling reduced by a comparable VEGF inhibitor and a comparable Ang2 inhibitor encoded from two different non-naturally occurring polynucleotides.

[0008]

[0008] In some aspects, described herein is a viral vector comprising the non-naturally occurring polynucleotide described herein. In some embodiments, the viral vector is a scAAV vector. In some embodiments, the viral vector comprises an AAV serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

[0009]

[0009] In some aspects, described herein are cells that comprise the non-naturally occurring polynucleotides described herein. In some embodiments, the cells comprise embryonic stem cells, differentiated cells derived from embryonic stem cells, retinal pigment epithelial (RPE) cells, neural progenitor cells, photoreceptor progenitor cells, bone marrow derived hematopoietic stem cells, or bone marrow derived hematopoietic stem progenitor cells.

[0010]

[0010] In some aspects, described herein are pharmaceutical compositions comprising the non-naturally occurring polynucleotides or cells described herein. In some embodiments, the pharmaceutical compositions are formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intralateral cerebroventricularly, intraparenchymally, subcutaneously, intratumorally, pulmonarily, intratracheally, intraperitoneally, intravesically, intravaginally, rectally, orally, sublingually, transdermally, by inhalation, by inhalation spray form, by intracavitary-GI route, or combinations thereof. In some embodiments, the pharmaceutical composition is for treating an ocular disease or condition. In some embodiments, the pharmaceutical composition reduces neovascularization, vascular leakage, inflammation, or combinations thereof in a subject.

[0011]

[0011] In some aspects, described herein are methods for treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a non-naturally occurring polynucleotide, cell, or pharmaceutical composition described herein. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce neoangiogenic signaling when expressed in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neoangiogenic signaling in the absence of the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2. In some embodiments, the VEGF inhibitor and the activator of RTK / Tie2 or RTK / Tie2 reduce neoangiogenic signaling in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the neoangiogenic signaling reduced by a comparable VEGF inhibitor and a comparable activator of RTK / Tie2 or a comparable RTK / Tie2 encoded from two different non-naturally occurring polynucleotides. In some embodiments, the non-naturally occurring polynucleotide, cell or pharmaceutical composition described herein reduces neoangiogenesis, vascular leakage, inflammation, or a combination thereof in a subject. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce neovascularization in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neovascularization in the absence of the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2.In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce neovascularization in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neovascularization reduced by a comparable VEGF inhibitor and a comparable RTK / Tie2 activator or a comparable RTK / Tie2 encoded from two different non-naturally occurring polynucleotides. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce vascular leakage in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to vascular leakage in the absence of the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce vascular leakage in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the vascular leakage reduced by a comparable VEGF inhibitor and a comparable RTK / Tie2 activator or a comparable RTK / Tie2 encoded from two different non-naturally occurring polynucleotides.In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce inflammation in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to inflammation in the absence of the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2. In some embodiments, the VEGF inhibitor and the RTK / Tie2 activator or RTK / Tie2 reduce inflammation in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to inflammatory signaling reduced by a comparable VEGF inhibitor and a comparable RTK / Tie2 activator or a comparable RTK / Tie2 encoded from two different non-naturally occurring polynucleotides. In some embodiments, the disease or condition comprises ocular ischemia syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiency, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Malattia leventinese (familial dominant drusen), and blue-cone monochromacy. In some embodiments, the disease or condition comprises diabetic macular edema (DME). In some embodiments, the disease or condition comprises diabetic macular retinopathy (DMR).

[0012]

[0012] In some aspects, described herein is a kit comprising a non-naturally occurring polynucleotide, a cell, or a pharmaceutical composition described herein; and a container.

[0013] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the agency upon request and payment of the necessary fee. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 illustrates targets that can be inhibited or modulated by VEGF inhibitors, activators of RTK / Tie2 or RTK / Tie2 described herein to modulate or reduce neovascularization. [Diagram 2]

[0015] FIG. 2 illustrates an exemplary adeno-associated virus (AAV) vector comprising a non-naturally occurring polynucleotide described herein, where the non-naturally occurring polynucleotide comprises two expression cassettes encoding a combination of a VEGF inhibitor, including a VEGF antibody, and either an Ang1 fragment or an Ang2 shRNA. [Figure 3A]

[0016] FIG. 3A illustrates the expression levels (μg / ml) of endogenous Ang2 inhibited by a non-antibody VEGF inhibitor (aflibercept or VEGF-Trap) encoded by an exemplary AAV vector comprising a non-naturally occurring polynucleotide described herein (AAV2.N54-120-136 or AAV2.N54-120-153) and an Ang1 fragment (Ang1-FLD) or an Ang2 shRNA encoded by a different exemplary AAV vector (AAV2.N54-120-150 or AAV2.N54-120-148). [Figure 3B]

[0017] FIG. 3B illustrates additional AAV vectors in which the RTK / Tie2 activator comprises either an Ang1 fragment (Ang1-FOLD or Ang1-FTD) or an Ang2 shRNA. [Figure 4]

[0018] FIG. 4 illustrates the expression levels (μg / ml) of endogenous Ang2 inhibited by a non-antibody VEGF inhibitor (aflibercept or VEGF-Trap) and an Ang2 shRNA, where the non-antibody VEGF inhibitor and the Ang2 shRNA were encoded by an exemplary AAV vector described herein (AAV2.N54-120-150 or AAV2.N54-120-148). [Diagram 5]

[0019] FIG. 5 illustrates the expression levels (μg / ml) of endogenous Ang2 inhibited by a VEGF inhibitor (VEGF-scFv antibody) and an Ang2 shRNA, where the VEGF antibody and the Ang2 shRNA were encoded by an exemplary AAV vector described herein. [Figure 6A]

[0020] FIG. 6A illustrates an exemplary pFB-AAV vector map comprising at least two expression cassettes described herein, where the AAV vector illustrated is a baculovirus-based AAV vector. [Figure 6B]

[0021] Figure 6B illustrates an exemplary pFB-AAV vector (AVMX103: anti-VEGF-(Fab)2-hCOMP-Ang1) containing a VEGF antibody (upper box, SEQ ID NO:21 and SEQ ID NO:22), hCOMP-Ang1 (middle box, SEQ ID NO:6) or FLAG-hCOMP-Ang1 (lower box, SEQ ID NO:8). [Figure 7A]

[0022] FIG. 7A illustrates an exemplary pFB-AAV vector (AVMX103: anti-VEGF-(Fab)2-hCOMP-Ang1). [Figure 7B]

[0023] 7B illustrates an exemplary pFB-AAV vector (AVMX103:VEGF(Fab)2-linker-hCOMP-Ang-1) for expressing a VEGF antibody fused to an Ang1 fragment. The heavy chain of the VEGF antibody is fused to a soluble polypeptide (hCOMP, SEQ ID NO:2) and an Ang1 fragment (SEQ ID NO:5) via a GGGGSG linker (upper box, SEQ ID NO:41), while the light chain of the anti-VEGF antibody is transcribed separately by a different expression cassette (lower box, SEQ ID NO:43). [Figure 7C]

[0024] 7C illustrates an exemplary pFB-AAV vector (AVMX103b:anti-VEGF-(Fab)2-linker-hCOMP-An AVMX103:VEGF(Fab)2-linker-Ang-1) containing a VEGF antibody fused to an Ang1 fragment (top box, SEQ ID NO:42 and bottom box, SEQ ID NO:43). The heavy chain of the VEGF antibody is fused to the Ang1 fragment (SEQ ID NO:5) via a GGGGSG linker (top box, SEQ ID NO:42), while the light chain of the anti-VEGF antibody is transcribed separately (bottom box, SEQ ID NO:43). [Figure 7D]

[0025] Figure 7D illustrates a non-limiting exemplary pFB-AAV vector (AVMX-110:VEGF-Trap-hCOMP-Ang1) encoding a non-antibody VEGF inhibitor (SEQ ID NO:24) and an Ang1 fragment (SEQ ID NO:6). [Figure 8A]

[0026] Figure 8A illustrates a non-limiting exemplary pFB-AAV vector (AVMX110-hCOMP-Ang1.FLD) encoding a non-antibody VEGF inhibitor (SEQ ID NO:24) fused to an Ang1 fragment (SEQ ID NO:6) via a linker designated "4xGGGGS." [Figure 8B]

[0027] Figure 8B illustrates a non-limiting exemplary pFB-AAV vector (AVMX110-Ang2-Antibody) encoding a non-antibody VEGF inhibitor (VEGF-Trap, SEQ ID NO:24) and an ANG2 antibody (SEQ ID NO:27). [Figure 8C]

[0028] FIG. 8C illustrates a non-limiting exemplary pFB-AAV vector encoding an scFv antibody VEGF inhibitor (SEQ ID NO: 23) linked (indicated by "4xGGGGS") to an ANG2 antibody (SEQ ID NO: 27). [Figure 8D]

[0029] Figure 8D illustrates a non-limiting exemplary pFB-AAV vector (AVMX110-anti-Ang2 shRNA) encoding a non-antibody VEGF inhibitor (VEGF-Trap, SEQ ID NO:24) and an ANG2 silencing sequence (e.g., a nucleic acid sequence encoding an inhibitory RNA). [Figure 9A]

[0030] Figure 9A illustrates a non-limiting exemplary pFB-AAV vector (AVMX104: anti-VEGF-ScFV-hCOMP-Ang1) encoding an antibody VEGF inhibitor (sequence number 34) and either hCOMP-Ang1 (for therapeutic purposes, sequence number 6) or FLAG-hCOMP-Ang1 (for pharmacokinetic purposes, sequence number 28). [Figure 9B]

[0031] 9B illustrates a non-limiting exemplary pFB-AAV vector (AVMX105:Flt1-D2 / KDR-D2) encoding a soluble VEGF inhibitor (SEQ ID NO:25). The AAV vector can include at least one more expression cassette for expressing any one of the VEGF inhibitors, activators of RTK / Tie2, or RTK / Tie2 described herein. [Figure 9C]

[0032] Figure 9C illustrates a non-limiting exemplary pFB-AAV vector (AVMX106:Flt1-D2 / KDR-D2-COMP-Ang1) encoding a soluble VEGF inhibitor (SEQ ID NO:25) and hCOMP-Ang1. [Figure 9D]

[0033] Figure 9D illustrates a non-limiting exemplary pFB-AAV vector encoding a soluble VEGF inhibitor (SEQ ID NO:25, upper box or SEQ ID NO:26, lower box) and hCOMP-Ang1 (Flt1-D2 / KDR-D2-hCOMP-Ang1). [Figure 9E]

[0034] 9E illustrates a non-limiting exemplary pFB-AAV vector (AVMX108:VEGF-scFv) encoding an scFv antibody VEGF inhibitor (SEQ ID NO: 35). The AAV vector can include at least one more expression cassette for expressing any one of the VEGF inhibitors, activators of RTK / Tie2, or RTK / Tie2 described herein. [Figure 9F]

[0035] FIG. 9F illustrates a non-limiting exemplary pFB-AAV vector encoding an scFv antibody VEGF inhibitor (SEQ ID NO:35) and hCOMP-Ang1 (VEGF-ScFV-hCOMP-Ang1). [Figure 10A]

[0036] FIG. 10A illustrates exemplary information regarding Ang1, COMP-Ang1, and the disadvantages of full-length Ang1. [Figure 10B]

[0037] FIG. 10B illustrates an exemplary dual expression AAV construct. [Figure 11]

[0038] FIG. 11 illustrates the dual gene construct compared to the single gene construct. [Figure 12]

[0039] FIG. 12 illustrates that VEGF promoted leakage, whereas aflibercept and Ang1 acted to reduce leakage of FITC-dextran. [Figure 13]

[0040] 13A and 13B show the results of Ang1 and VEGF-Trap construct comparisons as bar graphs±SEM, and statistical analysis using one-way ANOVA and multiple comparisons using Dunnett's test. [Figure 14]

[0041] FIG. 14 illustrates representative FA images from different groups. [Figure 15]

[0042] Figure 15 illustrates VEGF-Trap concentrations expressed in pg of aflibercept per eyecup. The eyecup consisted of the retina, sclera, choroid and retina. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015]

[0043] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

[0016] Overview

[0044] Abnormal expression of VEGF results in the pathogenesis of retinal tissue, such as neovascular age-related macular degeneration (nAMD), diabetic retinopathy (DMR), polypoidal choroidal vasculopathy (PCV), etc. Besides VEGF, many other factors, such as placenta-derived growth factor-B (PDGF-B), stromal cell-derived factor-1 (SDF-1), hypoxia-inducible factor-1 (HIF-1), receptor tyrosine kinase (RTK / Tie2), vascular cell adhesion molecule 1 (VCAM-1), neuropilin-1 (NP-1), neuropilin-2 (NP-2), ephrin or Eph (erythropoietin-producing hepatocellular carcinoma), have been found to be associated with neovascularization.

[0017]

[0045] The receptor tyrosine kinase TEK tyrosine kinase 2 (RTK / Tie2) and its associated ligands, angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2), are the most relevant factors responsible for the assembly and disassembly of the endothelial lining of blood vessels. Angiopoietins are involved in the control of microvascular permeability, vasodilation and vasoconstriction by signaling smooth muscle cells, pericytes and surrounding blood vessels. Ang1 is a physiological angiogenesis promoter in embryonic development and is produced by vascular smooth muscle cells. Ang1 function is essential for endothelial cell survival, vascular branching and pericyte recruitment. Ang1 is a glycoprotein of 498 amino acid residues with two isoforms, and a single amino acid mutation at glycine 269 (G269) is lost in isoform 2. The functional regions of Ang1, aa1-19, are the secretory signaling sequence (S); aa20-158 are the superclustering domain (SCD); aa159-255 are the coiled-coil oligomer domain (CCOD), and aa256--83:aa284-498 are the fibrinogen-like domain (FLD), RTK / Tie2 binding domain. Ang1 promotes the formation and maturation of tissue blood vessels and retinal vascular networks in postnatal development. Experimentally induced elevation of Ang1 can cause reduction of retinal vascular leukocyte adhesion, endothelial cell injury and blood-retinal barrier breakdown in diabetic retinopathy models, suppressed the development of CNV after laser wounding, and inhibited VEGF-mediated breakdown of the blood-retinal barrier in response to ischemia. The C-terminal FLD of Ang1 can dimerize and bind to RTK / Tie2 when fused at its N-terminus to a dimerization unit of a human unnamed protein sequence (designated hCOMP or Ang1-FLD dimerization unit) that aligns with the coiled-coil domain of rat cartilage oligomeric matrix protein (COMP), a 45 amino acid peptide. Ang2 is a growth factor that belongs to the angiopoietin / Tie (tyrosine kinase with Ig and EGF homology domains) signaling pathway, one of the main pathways involved in angiogenesis.Ang2 was identified by cDNA library screening shortly after the identification of ANG1, a potent angiogenic factor. Ang2 is critical for in vivo angiogenesis. Ang2, a 496 amino acid long protein, shares approximately 60% amino acid homology with Ang1 and lacks one of the nine cysteines found in mature ANG1. It has a secretory signaling peptide, an NH2-terminal coiled-coil domain, and a COOH-terminal fibrinogen-like domain. Unlike Ang1, Ang2 acts in an autocrine manner and its expression is highly regulated. Like Ang1, Ang2 binds to the Tie2 receptor with the same binding affinity, inducing an antagonistic role against Ang1. Ang2 expression is induced by inflammatory mediators such as thrombin accumulation, hypoxia, or cancer. RTK / Tie2 can be activated by expressing Ang1 or a fragment thereof, or by expressing an inhibitor of Ang2 (e.g., an inhibitory RNA or an antibody targeting Ang2), which then reduces the neoangiogenic signal.

[0018]

[0046] In some cases, additional RTK / Tie2 may be expressed in cells. Expressing RTK / Tie2 increases the frequency with which Ang1 (e.g., endogenously expressed Ang1) contacts and activates RTK / Tie2. In such a scenario, expression of RTK / Tie2 asserts the same effect as expression of Ang1 or reduction of expression of Ang2.

[0019]

[0047] Neovascularization plays a key role in the tissue development and pathogenesis of many diseases, including ocular ischemic syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis or neovascular uveitis. The clinical efficacy of intravitreal (IVT) anti-VEGF drugs has been widely demonstrated as a benchmark treatment in several angiogenesis-driven eye diseases, including diabetic macular edema (DMR), neovascular age-related macular degeneration (nAMD). Pegaptanib, ranibizumab (Lucentis) and aflibercept (Eylea) have been approved for ocular use, while bevacizumab (Avastin) is widely used by ophthalmologists to treat "off-label" patients to reduce treatment costs.

[0020]

[0048] These drugs are active in the nanomolar to picomolar range, but have a short duration of efficacy. Patients are required to receive one dose every 4-6 weeks. Many of them are associated with neovascularization, and patients are reliant on monthly administration of one of these anti-VEGF therapies. Challenges with anti-VEGF treatment of these eye diseases are the short durability of bioavailability and frequent IVT administration of anti-VEGF drugs, thus causing great inconvenience and financial burden to patients. As IVT drugs are administered, anti-VEGF antagonists in the vitreous humor (VH) increase or decrease, which results in pathophysiological instability and visual changes. Thus, there remains a need for therapeutic agents that can increase the therapeutic effect of VEGF inhibition. There is also a need for therapeutic agents that can decrease neovascularization or neovascularization signals.

[0021]

[0049] To address these needs, a non-naturally occurring polynucleotide that functions as a single delivery vehicle and includes one or more expression cassettes encoding a VEGF inhibitor and an activator of RTK / Tie2 is described herein. Figure 2 illustrates a non-limiting example of an AAV vector that includes a non-naturally occurring polynucleotide for expressing a VEGF inhibitor (e.g., a VEGF antibody) and an activator of RTK / Tie2, including either an Ang1 fragment (Ang1-FLD, an agonist of RTK / Tie2); or an Ang2 shRNA to reduce endogenous Ang2 (antagonist of the RTK / Tie2 signaling pathway) expression.

[0022]

[0050] In some embodiments, the non-naturally occurring polynucleotide can be part of a viral vector, such as an AAV vector. By utilizing such an AAV vector with one or more expression cassettes, different combinations of VEGF inhibitors and RTK / Tie2 activators including Ang1 can be constructed, including but not limited to anti-VEGF antibodies including IgG, Fab, F(ab)'2 or scFv or fragments thereof and Ang1 full length protein; non-antibody VEGF inhibitors including soluble Flt1 VEGF binding domain and Ang1 full length or fragment protein (e.g., Ang1-FLD fused to hCOMP); or non-antibody VEGF inhibitors including soluble Flt1 and Flk1 VEGF binding domain and Ang1 full length or fragment protein. By using this approach, the VEGF signaling pathway is decreased by the VEGF inhibitor, while the level of Ang1 expression is increased, resulting in the activation of RTK / Tie2. RTK / Tie2 activation results in the proliferation of pericytes, which strengthens the blood vessels and reduces vascular leakage and inflammation associated with vascular leakage.

[0023]

[0051] Alternatively, instead of increasing Ang1 expression, RTK / Tie2 can be activated by antagonizing Ang2 expression. Ang2 antagonists can include, but are not limited to, antibodies or inhibitory RNAs that target Ang2. AAV vectors that contain one or more expression cassettes can be used to deliver VEGF inhibitors and antibodies or inhibitory RNAs (e.g., shRNAs) that target Ang2 into cells to inhibit VEGF while simultaneously activating RTK / Tie2.

[0024]

[0052] Also described herein is a method for treating a disease or condition using the non-naturally occurring polynucleotide described herein. The disease or condition is associated with increased neovascularization, which results in pathology such as corneal neovascularization, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, or choroidal neovascularization in a subject. In some cases, the non-naturally occurring polynucleotide described herein can produce a synergistic therapeutic effect for treating a disease or condition. For example, a subject treated with a vector (e.g., as a single delivery vehicle) can show reduced angiogenesis, neovascularization, vascular leakage, inflammation, or a combination thereof, compared to when the subject receives VEGF inhibitor treatment only; treatment for activating RTK / Tie2 only; or treatment for activating RTK / Tie2 by different modalities (e.g., at different times, by different routes, or by different delivery vehicles).

[0025] Non-naturally occurring polynucleotides

[0053] Described herein is a non-naturally occurring polynucleotide comprising one or more expression cassettes for expressing a VEGF inhibitor; and a RTK / Tie2 or RTK / Tie2 activator. The VEGF inhibitor and the RTK / Tie2 or RTK / Tie2 activator can modulate neoangiogenic signaling in cells. In some embodiments, the VEGF inhibitor and the RTK / Tie2 or RTK / Tie2 activator reduce neoangiogenic signaling in cells. In some embodiments, the neoangiogenic signaling comprises a signaling pathway associated with vasculogenesis, angiogenesis or arteriogenesis. In some embodiments, the neoangiogenic signaling comprises a VEGF signaling pathway or an angiopoietin signaling pathway. Figure 1 illustrates non-limiting examples of ligands and receptors involved in the VEGF signaling pathway and the angiopoietin signaling pathway. For example, the VEGF signaling pathway is modulated by multiple VEGF or VEGF isoforms that bind to multiple VEGF receptors, while the angiopoietin signaling pathway is primarily modulated by angiopoietins (Ang1, Ang2, Ang3 and Ang4) that bind to RTK / Tie2. Figure 1 also illustrates potential VEGF or angiopoietin signaling pathway targets that can be modulated by the VEGF inhibitors and RTK / Tie2 or RTK / Tie2 activators described herein to reduce neoangiogenic signaling.

[0026]

[0054] In some embodiments, the non-naturally occurring polynucleotide comprises an expression cassette for expressing a VEGF inhibitor and an RTK / Tie2 or an activator of RTK / Tie2 as one contiguous polypeptide that is cleavable into separate polypeptides including a VEGF inhibitor, an RTK / Tie2 or an activator of RTK / Tie2. In some embodiments, the contiguous polypeptide comprises a protease peptide sequence. In some embodiments, the protease peptide sequence is cleavable by a protease endogenously expressed in the cell. Non-limiting examples of proteases include serine endoproteases, aspartic acid endoproteases, cysteine ​​thiol endoproteases, metalloendoproteases, or glutamic acid and threonine endoproteases. In some embodiments, the protease peptide sequence is cleavable by a serine endoprotease. In some embodiments, the protease peptide sequence is cleavable by furin. In some embodiments, the contiguous polypeptide comprises a protease cleavable sequence. In some embodiments, the protease-cleavable sequence can be cleaved by any one of the proteases described herein, hi some embodiments, the protease-cleavable sequence can be cleaved by furin.

[0027]

[0055] In some embodiments, the consecutive polypeptides include a self-cleaving polypeptide sequence. In some embodiments, the self-cleaving polypeptide sequence includes a 2A self-cleaving peptide sequence. Non-limiting examples of 2A self-cleaving peptide sequences can include T2A, P2A, E2A, F2A, or combinations thereof. In some embodiments, the self-cleaving polypeptide sequence includes a F2A peptide sequence. In some embodiments, the consecutive polypeptides include a protease-cleavable sequence and a self-cleaving polypeptide sequence. For example, the consecutive polypeptides described herein can include a furin-F2A fusion polypeptide sequence.

[0028]

[0056] In some embodiments, the non-naturally occurring polynucleotide comprises at least 2, at least 3, at least 4, at least 5 or more expression cassettes for a VEGF inhibitor and an RTK / Tie2 or an activator of RTK / Tie2. In some embodiments, the non-naturally occurring polynucleotide comprises two expression cassettes. In some embodiments, the VEGF inhibitor and the RTK / Tie2 or an activator of RTK / Tie2 are each expressed from an expression cassette. In other cases, the VEGF inhibitor may be partially expressed as a fusion protein by one of the two expression cassettes, while the other expression cassette expresses the remaining part of the VEGF inhibitor. For example, FIG. 7B illustrates a VEGF antibody fused to an Ang1 fragment. In this example, the heavy chain of the VEGF antibody is fused to a soluble polypeptide (hCOMP, SEQ ID NO:2) and an Ang1 fragment (SEQ ID NO:5) via a GGGGSG linker (upper box, SEQ ID NO:41), while the light chain of the anti-VEGF antibody is transcribed separately by a different expression cassette (lower box, SEQ ID NO:43). Another example is Figure 7C, which illustrates another exemplary AAV vector, whereby the heavy chain of a VEGF antibody is fused to an Ang1 fragment (sequence number 5) via a GGGGSG linker (upper box, sequence number 41), while the light chain of an anti-VEGF antibody is transcribed separately by a different expression cassette (lower box, sequence number 43).

[0029]

[0057] In some embodiments, the expression cassette comprises one or more promoters or internal ribosome entry sites (IRES). In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette is under the expression control of a promoter. In some embodiments, the expression cassette can further exert expression control by at least one IRES. In such an arrangement, expression of the VEGF inhibitor and the RTK / Tie2 or RTK / Tie2 activator can be achieved by only one expression cassette.

[0030]

[0058] In some embodiments, the VEGF inhibitor comprises an antibody or a fragment thereof. In some embodiments, the VEGF antibody binds to VEGF and reduces angiogenesis signaling involving the VEGF signaling pathway. In some embodiments, the VEGF inhibitor is not an antibody. For example, the VEGF inhibitor described herein can comprise a VEGF receptor, a combination of VEGF receptors, or a fragment thereof, for binding to VEGF to inhibit or reduce the VEGF signaling pathway. The VEGF receptor can comprise VEGF receptor 1 (FLT1), VEGF receptor 2 (KDR / FLK1), VEGF receptor 3 (FLT4), a fragment thereof, or a combination thereof. In some embodiments, the VEGF receptor can be a soluble VEGF receptor. For example, the soluble VEGF receptor can comprise a soluble VEGFR1, a soluble VEGFR2, a soluble VEGFR3, a soluble fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of FLT1, KDR / FLK1, FLT4, a fragment thereof, or a combination thereof. In some embodiments, the non-antibody VEGF inhibitor comprises at least one of soluble FLT1, soluble KDR / FLK1, soluble FLT4, fragments thereof, or combinations thereof. In some embodiments, the non-antibody inhibitor VEGF comprises VEGF-Trap. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:31 (Table 11). In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is SEQ ID NO:24. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is SEQ ID NO:25. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is SEQ ID NO:26. In some embodiments, the non-antibody VEGF inhibitor comprises a polypeptide sequence that is SEQ ID NO:31.

[0031] [Table 1-1]

[0032] [Table 1-2]

[0033] [Table 1-3]

[0034] [Table 1-4]

[0035]

[0059] In some embodiments, the RTK / Tie2 activator comprises a polypeptide or polynucleotide for activating RTK / Tie2. In some embodiments, the RTK / Tie2 activator comprises a polypeptide comprising an inhibitor, such as an antibody or a fragment thereof. In some embodiments, the RTK / Tie2 activator comprises a polypeptide comprising a non-antibody inhibitor for activating RTK / Tie2. In some embodiments, the RTK / Tie2 activator comprises a polypeptide corresponding to a full-length protein. For example, the RTK / Tie2 activator can be a full-length angiopoietin. In some cases, instead of a full-length protein, a fragment of a protein can be utilized as the RTK / Tie2 activator. For example, instead of a full-length angiopoietin, a fragment of an angiopoietin can be utilized to activate RTK / Tie2.

[0036]

[0060] In some embodiments, the RTK / Tie2 activator comprises a polypeptide corresponding to a full-length protein. In some embodiments, the RTK / Tie2 activator comprises a polypeptide encoded from a full-length Ang1 or Ang2 nucleic acid sequence (SEQ ID NO:4 and SEQ ID NO:13, respectively, Table 12). In some embodiments, the RTK / Tie2 activator comprises a polypeptide corresponding to full-length Ang1 or Ang2 (SEQ ID NO:3 and SEQ ID NO:12, respectively, Table 12). In some embodiments, the RTK / Tie2 activator comprises a polypeptide comprising full-length Ang1. In some embodiments, the RTK / Tie2 activator comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3.

[0037] [Table 2-1]

[0038] [Table 2-2]

[0039] [Table 2-3]

[0040] [Table 2-4]

[0041]

[0061] In some embodiments, the activator of RTK / Tie2 comprises a polypeptide corresponding to a fragment of a full-length protein. In some embodiments, the activator of RTK / Tie2 comprises a polypeptide corresponding to a fragment of a full-length angiopoietin. In some embodiments, the activator of RTK / Tie2 comprises a polypeptide corresponding to a fragment of angiopoietin comprising a polypeptide sequence comprising a length of at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids or more. In some embodiments, the activator of RTK / Tie2 comprising a fragment of angiopoietin comprises a polypeptide encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:4 or SEQ ID NO:13. In some embodiments, the RTK / Tie2 activator comprising a fragment of angiopoietin comprises a polypeptide encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to Ang1 (SEQ ID NO: 4). In some embodiments, the RTK / Tie2 activator comprising a fragment of angiopoietin comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 3 or SEQ ID NO: 12.

[0042]

[0062] In some embodiments, the RTK / Tie2 activator comprises a fragment of Ang1. The fragment of Ang1 can comprise Ang1 amino acids 1-19, secreted signaling sequence (S); Ang1 aa20-158, superclustering domain (SCD); Ang1 aa159-255, coiled-coil oligomer domain (CCOD), aa256-83: and Ang1 aa284-498, fibrinogen-like domain (FLD), which is a functional domain that binds to RTK / Tie2. In some embodiments, the RTK / Tie2 activator comprises a FLD fragment (functional fragment) of Ang1 (SEQ ID NO:5). In some embodiments, the RTK / Tie2 activator comprises a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:5. In some embodiments, the activator of RTK / Tie2 comprises a VEGF inhibitor and a fragment of Ang1 fused to a soluble peptide to increase the solubility of RTK / Tie2 or the activator of RTK / Tie2. In some embodiments, the soluble peptide comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the soluble peptide comprises a polypeptide sequence that is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94% or at most 93% identical to SEQ ID NO:1. In some embodiments, the soluble peptide comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:2. In some embodiments, the soluble peptide comprises a polypeptide sequence of SEQ ID NO:2. In some embodiments, a peptide tag such as a FLAG tag (SEQ ID NO: 10, encoded from the nucleic acid sequence of SEQ ID NO: 11) may be added to the FLD fusion. The additional FLAG tag may be used for pharmacokinetic purposes and measurements.In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:6. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide sequence of SEQ ID NO:6. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:7. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide encoded from a nucleic acid sequence of SEQ ID NO:7. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:8. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide sequence of SEQ ID NO:8. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide encoded from a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:9. In some embodiments, the RTK / Tie2 activator comprising a fragment of Ang1 is fused to a soluble peptide comprising a polypeptide encoded from a nucleic acid sequence of SEQ ID NO:9. Table 13 sets forth the nucleic acid and polypeptide sequences of variants of RTK / Tie2 activators, including FLD and soluble polypeptide fusions.

[0043] [Table 3-1]

[0044] [Table 3-2]

[0045] [Table 3-3]

[0046] antibody

[0063] In some embodiments, the VEGF inhibitor is a VEGF antibody. In some embodiments, the VEGF antibody comprises a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody, or a combination thereof, a binding fragment thereof, or a chemically modified derivative thereof.

[0047]

[0064] In some embodiments, the VEGF antibody binds to VEGF and reduces the VEGF signaling pathway. In some embodiments, the VEGF inhibitor, when delivered in combination with an RTK / Tie2 activator by a non-naturally occurring polynucleotide described herein, synergistically reduces the VEGF signaling pathway in a cell compared to the reduction in the VEGF signaling pathway induced by separate delivery of the VEGF inhibitor and the RTK / Tie2 activator (e.g., a VEGF inhibitor and a RTK / Tie2 activator delivered to a cell by two separate vectors) and / or by delivery of the VEGF inhibitor or the RTK / Tie2 activator alone. In some embodiments, a VEGF inhibitor, when delivered in combination with a RTK / Tie2 activator by a vector described herein, synergistically increases the RTK / Tie2 signaling pathway in a cell compared to the increase in the RTK / Tie2 signaling pathway induced by separate delivery of the VEGF inhibitor and the RTK / Tie2 activator (e.g., a VEGF inhibitor and a RTK / Tie2 activator delivered to a cell by two separate vectors) and / or by delivery of the VEGF inhibitor or the RTK / Tie2 activator alone.

[0048]

[0065] In some embodiments, the VEGF antibody binds to VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the VEGF antibody binds to one or more isoforms of VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, or VEGF206. In some embodiments, the antibody comprises IgG, Fab, Fa(ab)'2, single chain fragment variable (scFv), fragments thereof, or combinations thereof. Non-limiting examples of VEGF antibodies include ranibizumab or bevacizumab. In some embodiments, the VEGF antibody comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or a combination thereof or a fragment thereof (Table 13). In some embodiments, the VEGF antibody is an scFv antibody. In some embodiments, the VEGF scFv antibody comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:23, or a fragment thereof (Table 14).

[0049] [Table 4-1]

[0050] [Table 4-2]

[0051]

[0066] In some embodiments, the VEGF antibody comprises at least one heavy chain and at least one light chain. In such a scenario, at least one heavy chain and at least one light chain can be expressed separately by at least two expression cassettes. Moreover, the heavy chain or light chain can be further fused to any of the RTK / Tie2 activators described herein. For example, FIG. 7B illustrates a non-limiting example of a VEGF antibody fused to an Ang1 fragment. The heavy chain of the VEGF antibody is fused to a soluble polypeptide (hCOMP, SEQ ID NO:2) and an Ang1 fragment (SEQ ID NO:5) via a GGGGSG linker (upper box, SEQ ID NO:41), while the light chain of the anti-VEGF antibody is transcribed separately by a different expression cassette (lower box, SEQ ID NO:43). Another example is Figure 7C, which illustrates another exemplary AAV vector, whereby the heavy chain of a VEGF antibody is fused to an Ang1 fragment (sequence number 5) via a GGGGSG linker (upper box, sequence number 41), while the light chain of an anti-VEGF antibody is transcribed separately by a different expression cassette (lower box, sequence number 43).

[0052]

[0067] In some embodiments, the antibody encoded by the non-naturally occurring polynucleotide described herein is an activator of RTK / Tie2. In some embodiments, the antibody is an Ang2 antibody. In some embodiments, the binding of the Ang2 antibody reduces the VEGF signaling pathway described herein. In some embodiments, the binding of the Ang2 antibody to Ang2 synergistically reduces the VEGF signaling pathway in the presence of a VEGF inhibitor, compared to the reduction of the VEGF signaling pathway induced by the VEGF inhibitor alone or by the Ang2 antibody alone.

[0053]

[0068] In some embodiments, the Ang2 antibody binds to an Ang2 polypeptide or a fragment thereof encoded from a nucleic acid sequence or a fragment thereof that is least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 13 (Table 15). In some embodiments, the Ang2 antibody binds to an Ang2 polypeptide or a fragment thereof comprising a peptide sequence or a fragment thereof that is least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 12 (Table 15). In some embodiments, the Ang2 antibody comprises a polypeptide sequence that is least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or a fragment thereof or a combination thereof. In some embodiments, the Ang2 antibody comprises a polypeptide sequence that is SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the Ang2 antibody comprises a polypeptide sequence that is SEQ ID NO:27.

[0054] [Table 5-1]

[0055] [Table 5-2]

[0056]

[0069] In some embodiments, the antibody or antigen-binding fragment of the present disclosure includes variants or derivatives thereof. For example, non-human animals can be genetically modified to produce antibody variants or derivatives. In some embodiments, the antibody can be a single domain antibody (sdAb), such as a heavy chain only antibody (HCAb), a VHH, or a nanobody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fab IL-6R dimers and trimers, Fv, scFv, minibodies, dia-, tria-, and tetrabodies, and linear antibodies. Fab and Fab' are antigen-binding fragments that include the VH and CH1 domains of the heavy chain linked to the VL and CL domains of the light chain by disulfide bonds. F(ab')2 includes two Fabs or Fab's linked by disulfide bonds. Fv includes the VH and VL domains held together by non-covalent interactions. scFv (single chain variable fragment) is a fusion protein that comprises VH and VL domains connected by a peptide linker. Manipulation of the orientation of VH and VL domains and the length of the linker can be used to create molecules with different forms that can be monomers, dimers (diabodies), trimers (triabodies) or tetramers (tetrabodies). Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0057]

[0070] In some embodiments, the antibody is a binding fragment thereof. In some cases, the antibody is a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a multispecific antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a single domain antibody (e.g., a Nanobody®) thereof. In some embodiments, the antibody may be a multispecific antibody. In some cases, the multispecific antibody comprises two or more target binding moieties, each of which specifically binds to an antigen, and the two or more antigens are different. In some cases, the multispecific antibody comprises a target binding moiety that specifically binds to three or more different antigens, four or more different antigens, or five or more different antigens. In some embodiments, the antibody may be a bispecific antibody. In some cases, the bispecific antibody or binding fragment is referred to as Knobs-into-Holes (KiH), Asymmetric Re-engineering Technology-immunoglobulin (ART-Ig), Triomab quadroma, Bispecific Monoclonal Antibody (BiMAb, BsmAb, BsAb, bsMab, BS-Mab or Bi-MAb), FcΔAdp, XmAb, Azymetric, Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT), Bispecific T-cell Engager (BiTE), Biclonics, Fab-scFv-Fc, Two-in-one / Dual Action These include Fab (DAF), FinomAb, scFv-Fc-(Fab) fusions, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), Tandem diAbody (TandAb), Dual-sffinity-ReTargeting (DART) or nanobodies.

[0058]

[0071] In some embodiments, the antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the antibodies comprise an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the antibodies comprise an IgG1 framework. In some cases, the antibodies comprise an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the antibodies comprise an IgG2a framework. In some cases, the antibodies comprise an IgG2b framework. In some cases, the antibodies comprise an IgG3 framework. In some cases, the antibodies comprise an IgG4 framework.

[0059]

[0072] In some cases, the antibody described herein comprises one or more mutations in the framework region, for example, in the CH1 domain, the CH2 domain, the CH3 domain, the hinge region, or a combination thereof. In some cases, the one or more mutations are for stabilizing the antibody and / or increasing half-life. In some cases, the one or more mutations are for modulating Fc receptor interaction, reducing or eliminating Fc effector function, for example, FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In additional cases, the one or more mutations are for modulating glycosylation. Inhibitory RNA

[0073] In some embodiments, the RTK / Tie2 activators described herein include RNA or DNA. In some cases, the RTK / Tie2 activators include inhibitory RNA for modulating the signaling pathway by decreasing the expression of the protein. In some embodiments, the inhibitory RNA targets and decreases the expression of VEGF. In some embodiments, the inhibitory RNA targets and decreases the expression of angiopoietin. In some embodiments, the inhibitory RNA targets and decreases the expression of Ang2, resulting in an increase in the RTK / Tie2 signaling pathway. The inhibitory RNA can target and bind to the nucleic acid sequence of Ang2. In some embodiments, the inhibitory RNA targets and binds to the Ang2 transcript that includes a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 13. In some examples, the RNA comprises small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) or heterogeneous nuclear RNA (hnRNA). In some examples, the RNA comprises shRNA. In some examples, the RNA comprises miRNA. In some examples, the RNA comprises dsRNA. In some examples, the RNA comprises tRNA. In some examples, the RNA comprises rRNA. In some examples, the RNA comprises hnRNA. In some examples, the RNA comprises siRNA. In some examples, the signaling regulator comprises shRNA.

[0060]

[0074] In some embodiments, the RTK / Tie2 activator, including the inhibitory RNA, is about 10 to about 50 nucleotides in length. In some examples, the signaling transduction regulator is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length. In some embodiments, the signaling transduction regulator hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive bases of a target sequence described herein.

[0061]

[0075] In some embodiments, the activator of RTK / Tie2 comprises an shRNA for targeting and reducing endogenous expression of Ang2. Figure 3B, Figure 5 and Table 10 illustrate the inhibitory effect of Ang2 shRNA on endogenous Ang2. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to any one of SEQ ID NOs: 81-86 (Table 16). In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is any one of SEQ ID NOs: 81-86. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 81. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 82. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 83. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 84. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 85. In some embodiments, the Ang2 shRNA comprises a nucleic acid sequence that is SEQ ID NO: 86. In some embodiments, the Ang2 shRNA does not comprise a nucleic acid sequence that is SEQ ID NO:87.

[0062] [Table 6]

[0063] method Vector construction and delivery

[0076] Described herein is a method for generating a non-naturally occurring polynucleotide comprising one or more expression cassettes. In some embodiments, the non-naturally occurring polynucleotide is part of an AAV vector. In some embodiments, the non-naturally occurring polynucleotide comprises one or more promoters or IRES. Figures 6-9 and Table 17 illustrate exemplary AAV vectors showing non-naturally occurring polynucleotides comprising the arrangement of one or more expression cassettes.

[0064] [Table 7-1]

[0065] [Table 7-2]

[0066] [Table 7-3]

[0067]

[0077] Non-naturally occurring polynucleotides can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art.For example, non-naturally occurring polynucleotides can be transferred into host cells by physical, chemical or biological means.In some embodiments, non-naturally occurring polynucleotides can be delivered into cells by physical methods such as calcium phosphate precipitation, lipofection, particle gun, microinjection, gene gun, electroporation, etc.

[0068]

[0078] Physical methods for introducing a non-naturally occurring encoding polynucleotide into a cell can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, etc. One method for the introduction of a non-naturally occurring polynucleotide into a host cell is calcium phosphate transfection.

[0069]

[0079] Chemical means for introducing non-naturally encoding non-naturally occurring polynucleotides into cells can include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes or lipid nanoparticles. An example of a colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of non-naturally occurring polynucleotides or vectors encoding non-naturally occurring polynucleotides by targeted nanoparticles.

[0070]

[0080] When a non-viral delivery system is utilized, an example of a delivery vehicle is a liposome. The use of lipid formulations is considered for the introduction of non-naturally occurring polynucleotides or vectors encoding non-naturally occurring polynucleotides into cells (in vitro, ex vivo or in vivo). In another aspect, the vector can be associated with lipids. The lipid-associated vector can be encapsulated in the aqueous interior of the liposome, can be interspersed within the lipid bilayer of the liposome, can be attached to the liposome via a linking molecule associated with both the liposome and the non-naturally occurring polynucleotide, can be entrapped within the liposome, can be complexed with the liposome, can be dispersed in a solution containing lipids, can be mixed with lipids, can be combined with lipids, can be contained as a suspension in lipids, can be contained in or complexed with micelles, or can be otherwise associated with lipids. The lipid, lipid / DNA or lipid / expression vector association compositions are not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, as micelles or with a "collapsed" structure. Instead, they simply disperse in the solution, forming aggregates that are likely not uniform in size or shape.Lipids are fatty substances that, in some embodiments, are naturally occurring or synthetic lipids.For example, lipids include the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols and aldehydes, along with lipid droplets that naturally occur in cytoplasm.

[0071]

[0081] Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are often stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of single and multilamellar lipid vehicles formed by the creation of encased lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions with structures that differ from the normal vesicular structure in solution are also encompassed. For example, lipids in some embodiments assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0072]

[0082] In some cases, the non-viral delivery method includes lipofection, nucleofection, microinjection, biolistec, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced incorporation of polypeptides or DNA. In some embodiments, the delivery method includes conjugating or encapsulating the compositions described herein or non-naturally occurring polynucleotides with at least one polymer, such as a natural polymer or a synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, grafted) of two or more of several different monomers, such as lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc. In one example, the scaffold can be composed of a polymer that includes glycolic acid and lactic acid, such as one having a 90 / 10 or 5 / 95 ratio of glycolic acid to lactic acid. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragment(s) derived from these components, elastin, laminin, decorin, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0073]

[0083] In some cases, the non-naturally occurring polynucleotides described herein can be packaged and delivered to cells via extracellular vesicles. The extracellular vesicles can be any membrane-bound particle. In some embodiments, the extracellular vesicles can be any membrane-bound particle secreted by at least one cell. In some cases, the extracellular vesicles can be any membrane-bound particle synthesized in vitro. In some cases, the extracellular vesicles can be any membrane-bound particle synthesized without cells. In some cases, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomers, enveloped viruses, exomeres or other very large extracellular vesicles.

[0074]

[0084] In some embodiments, non-naturally occurring polynucleotides can be delivered into cells by biological methods, such as the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV vectors), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some examples, retroviral vectors include gamma-retroviral vectors, such as vectors derived from the Moloney murine leukemia (Keukemia) virus (MoMLV, MMLV, MuLV, or MLV) or mouse stem (Steam) cell virus (MSCV) genomes. In some examples, retroviral vectors also include lentiviral vectors, such as those derived from the human immunodeficiency virus (HIV) genome. In some examples, AAV comprises serotypes including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or combinations thereof. Based on these initial serotypes, the AAV capsid of each serotype can be engineered to be better suited for biological function, tissue or cell selection. In some embodiments, AAV is AAV2 and variants AAV2.N53 and AAV2.N54 used in the examples of the present invention. Chimeric AAVs that can contain at least two AAV serotypes are also considered. In some cases, at least 3, at least 4, at least 5, at least 6, at least 7 or up to 8 different serotypes are combined in the chimeric AAV. In some cases, only a portion of the AAV is chimeric. For example, suitable portions can include capsid, VP1, VP2 or VP3 domains and / or Rep.In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to an otherwise comparable wild-type AAV capsid protein. In some cases, the mutations can occur in VP1 and VP2, in VP1 and VP3, in VP2 and VP3, or in VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, such as about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some cases, the VPs can be removed. For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0075]

[0085] In some examples, the viral vector is a chimeric viral vector that includes viral portions from two or more viruses. In additional examples, the viral vector is a recombinant viral vector. In some cases, the vector includes additional features. Additional features can include sequences such as tags, signaling peptides, intron sequences, promoters, stuffer sequences, and the like. In some cases, the vector includes a signaling peptide. A signaling peptide, sometimes referred to as a signaling sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide, is a short peptide that is present at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway. Such proteins include proteins that reside inside certain organelles (endoplasmic reticulum, Golgi, or endosomes), are secreted from the cell, or are inserted into many cell membranes. In some cases, the nucleic acids provided herein can include a signaling peptide. A signaling peptide can be of any length, but is typically 15-30 amino acids in length. The signaling peptide can be about 10-15, 10-20, 10-30, 15-20, 15-25, 15-30, 20-30, or 25-30 amino acids in length. A variety of signaling peptides can be utilized, including, but not limited to, human antibody heavy chain (Vh), human antibody light chain (Vl), and aflibercept.

[0076]

[0086] In certain embodiments, additional features of the vector include a promoter. A promoter is a sequence of DNA to which a protein binds that initiates transcription of a single RNA from the DNA downstream of the promoter. The RNA can code for a protein or can have a function in itself, such as tRNA, mRNA, or rRNA. The promoter is located upstream in the DNA (towards the 5' region of the sense strand), near the transcription start site of the gene. The promoter can be about 100-1000 base pairs in length. In some cases, the promoter can be an inducible promoter. A variety of promoters are contemplated and may be used in the vectors of the present disclosure. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a simian vacuolating virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human beta actin promoter, a CAG promoter, a tetracycline response element (TRE) promoter, a UAS promoter, an actin 5c (Ac5) promoter, a polyhedron promoter, a Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GAL1 promoter, a GAL10 promoter, a TEF1 promoter, a glyceraldehyde 3-phosphage dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, a human polymerase III RNA (H1) promoter, a U6 promoter, a polyadenylation construct thereof, and any combination thereof. In some cases, the promoter is a CMV promoter.

[0077]

[0087] In some embodiments, the vector comprises at least two expression cassettes under the expression control of two different promoters. Such an arrangement allows two signaling transduction regulators to be expressed simultaneously or in a desired sequential order in cells. For example, a vector comprising a VEGF inhibitor and an Ang1 protein can be engineered to constitutively express the VEGF inhibitor (e.g., the VEGF inhibitor is under the control of a CMV promoter), while the Ang1 protein can be expressed at a later time point (e.g., the Ang1 protein is under the control of an inducible promoter). In some cases, the use of two promoters also allows the expression of two signaling transduction regulators to be modulated. For example, the VEGF inhibitor can be driven by a promoter with strong expression activity in a specific cell type, while the Ang1 protein is driven by a different promoter with weaker expression activity in the same cell type.

[0078]

[0088] In some embodiments, methods of modifying cells to generate engineered cells are also provided herein. The cells can refer to primary cells, recombinant cells, or cell lines. In some cases, the cells are packaging cells. The packaging cells can be any one of HEK293 cells, HeLa cells, and Vero cells, to name a few. The engineered cells can be primary cells. In some cases, the engineered cells can be ocular cells. Suitable ocular cells include, but are not limited to, photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and others.

[0079]

[0089] In some cases, the cell is a packaging cell that is used to generate viral particles.To generate AAV virions or viral particles, AAV vectors are introduced into suitable host cells using known techniques, such as by transfection.In some cases, transfection techniques are used, such as CaPO4 transfection or electroporation, and / or hybrid adenovirus / AAV vector infection into cell lines, such as human embryonic kidney cell line HEK293 (a human kidney cell line that contains a functional adenovirus E1 gene that provides trans-acting E1 protein).Suitable transfection methods include calcium phosphate co-precipitation, direct microinjection, electroporation, liposome-mediated gene transfer, and high-velocity microprojectile-based nucleic acid delivery, as known in the art.

[0080]

[0090] To engineer the cells, a plurality of cells may be contacted with the isolated non-naturally occurring nucleic acid. The contact can include any length of time, including from about 5 minutes to about 5 days. The contact can last for about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes. In some cases, the contact can last for 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, or up to about 5 days.

[0081]

[0091] In some cases, the packaging cell line supernatant is treated by PEG precipitation to concentrate the virus. In other cases, a centrifugation step can be used to concentrate the virus. For example, a column can be used to concentrate the virus during centrifugation. In some embodiments, the precipitation is carried out at about 4° C. or below (e.g., about 3° C., about 2° C., about 1° C., or about 1° C.) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some embodiments, the recombinant AAV is isolated from the PEG-precipitated supernatant by low-speed centrifugation followed by a CsCl gradient. The low-speed centrifugation can be at about 4000 rpm, about 4500 rpm, about 5000 rpm, or about 6000 rpm for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some cases, recombinant AAV is isolated from the PEG-precipitated supernatant by centrifugation at about 5000 rpm for about 30 minutes followed by CsCl gradient. In some cases, CsCl purification can be replaced by IDX gradient ultracentrifugation. Supernatant can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours after transfection or at any time between the two time points mentioned above. Supernatant can be purified, concentrated or a combination thereof. For example, concentration or viral titer can be determined by qPCR or silver staining.

[0082]

[0092] In one embodiment, a plurality of AAV particles (containing the non-naturally occurring polynucleotides described herein) isolated from the engineered cells is also provided. The viral titer is about 10 2 vp / mL, approx. 10 3 vp / mL, approx. 10 4 vp / mL, approx. 10 5 vp / mL, approx. 10 6 vp / mL, approx. 10 7 vp / mL, approx. 10 8 vp / mL or up to about 10 9 The viral titer can be approximately 10 2GC / mL, approx. 10 3 GC / mL, approx. 10 4 GC / mL, approx. 10 5 GC / mL, approx. 10 6 GC / mL, approx. 10 7 GC / mL, approx. 10 8 GC / mL or up to about 10 9 In some cases, the viral titer may be about 10 2 TU / mL, approximately 10 3 TU / mL, approximately 10 4 TU / mL, approximately 10 5 TU / mL, approximately 10 6 TU / mL, approximately 10 7 TU / mL, 10 8 TU / mL or up to about 10 9 The optimal viral titer may be in TU / mL. Optimal viral titers may vary depending on the cell type to be transduced. The viral range may be about 1000 MOI to about 2000 MOI, about 1500 MOI to about 2500 MOI, about 2000 MOI to about 3000 MOI, about 3000 MOI to about 4000 MOI, about 4000 MOI to about 5000 MOI, about 5000 MOI to about 6000 MOI, about 6000 MOI to about 7000 MOI, about 7000 MOI to about 8000 MOI, about 8000 MOI to about 9000 MOI, about 9000 MOI to about 10,000 MOI. For example, to infect one million cells using an MOI of 10,000, it would be 10,000 x 1,000,000 = 10 10 A GC would be required.

[0083]

[0093] In some cases, multiple AAV particles can be formulated into a unit dose form. Various formulations are contemplated for adult or pediatric delivery, ranging from 0.5×10 9 vg, 1.0×10 9 vg, 1.0×10 10 , 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14vg, 1.0×10 15 vg or up to 1.5×10 15 The composition of viral particles, including but not limited to, .vg, may be frozen or otherwise stored in a suitable container.

[0084]

[0094] The compositions and methods provided herein may be sufficient to enhance delivery and / or expression of a subject biologic by at least about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% more than an otherwise comparable unmodified nucleic acid. In some cases, the otherwise comparable unmodified nucleic acid is a nucleic acid encoding VEGF-Trap. In some cases, the modification is at least about 1 fold, about 6 fold, about 11 fold, about 16 fold, about 21 fold, about 26 fold, about 31 fold, about 36 fold, about 41 fold, about 46 fold, about 51 fold, about 56 fold, about 61 fold, about 66 fold, about 71 fold, about 76 fold, about 81 fold, about 86 fold, about 91 fold, about 96 fold, about 101 fold, about 106 fold, about 111 fold, about 116 fold, about 121 fold, about 126 fold, about 131 fold, about 136 fold, about 141 fold, about 146 fold, about 151 fold, about 156 fold, about 161 fold, about 166 fold, about 171 fold, about 176 fold, about 181 fold, about 186 fold, about 187 fold, about 189 fold, about 190 fold, about 192 fold, about 194 fold, about 196 fold, about 198 fold, about 199 fold, about 200 fold, about 201 fold, about 202 fold, about 203 fold, about 204 fold, about 205 fold, about 206 fold, about 207 fold, about 208 fold, about 209 fold, about 210 fold, about 211 fold, about 212 fold, about 214 fold, about 216 fold, about 218 fold, about 219 fold, about 220 fold, about 225 fold, about 22 6-fold, about 191-fold, about 196-fold, about 201-fold, about 206-fold, about 211-fold, about 216-fold, about 221-fold, about 226-fold, about 231-fold, about 236-fold, about 241-fold, about 246-fold, about 251-fold, about 256-fold, about 261-fold, about 266-fold, about 271-fold, about 276-fold, about 281-fold, about 286-fold, about 291-fold, about 296-fold, about 301-fold, about 306-fold, about 311-fold, about 316-fold, about 321-fold, about 326-fold, about 331-fold, about 336-fold, about 341-fold, about 346-fold or about 350-fold more may be sufficient to enhance delivery and / or expression of a subject biologic. In certain embodiments, increased expression comprises at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, at least a 100-fold, at least a 200-fold or at least a 500-fold increase as determined by an in vitro assay. Suitable in vitro assays include ELISA, Western blot, Luminex, microscopy, imaging and / or flow cytometry.

[0085]

[0095] The subject AAV virions can exhibit at least 1-fold, at least 6-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased infectivity of retinal cells (photoreceptors, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, etc.) compared to the infectivity of retinal cells by AAV virions containing otherwise comparable WT AAV capsid proteins.

[0086] treatment

[0096] Provided herein is a method for treating a disease or condition described herein. The treatment method can include introducing a non-naturally occurring polynucleotide, an AAV vector comprising a non-naturally occurring polynucleotide, or an AAV comprising a non-naturally occurring polynucleotide into a subject in need thereof. Also provided is a method for treating a disease or condition, comprising administering a pharmaceutical composition to a subject in need thereof. The pharmaceutical composition can include a sequence encoding a biologic comprising a non-naturally occurring polynucleotide, an AAV vector comprising a non-naturally occurring polynucleotide, or an AAV comprising a non-naturally occurring polynucleotide. In some embodiments, administration is by any suitable administration mechanism, including systemic administration (e.g., intravenous, inhalation, intravitreal, etc.). In some embodiments, the subject is a human.

[0087]

[0097] In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition is administered at least once during a period of time (e.g., every 2 days, twice a week, once a week, every week, three times a month, twice a month, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered more than once during a period of time (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times).

[0088]

[0098] In some embodiments, the method includes administering a non-naturally occurring polynucleotide, an AAV vector comprising a non-naturally occurring polynucleotide, an AAV comprising a non-naturally occurring polynucleotide, or a pharmaceutical composition in a therapeutically effective amount by various forms and routes, including, for example, oral or topical administration. In some embodiments, the composition can be administered parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, intrathecally, intraocularly, intrasternally, ophthalmic, endodermal, topically, intranasally, intrapulmonary, rectally, intraarterially, intrathecally, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, such as by injection or infusion. In some embodiments, the composition can be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa administration). In some embodiments, the composition is delivered by multiple routes of administration.

[0089]

[0099] In some embodiments, the method comprises administering the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition by intravenous infusion. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition is administered by slow continuous infusion over a long period of time, such as more than 24 hours. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition is administered as an intravenous injection or short infusion. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition is administered by intravitreal route. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition can be administered in a localized manner, for example, by injection of the agent directly into an organ, optionally in a depot or sustained release formulation or implant.

[0090]

[0100] In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition may be administered in conjunction with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, the non-naturally occurring polynucleotide or the pharmaceutical composition comprising the non-naturally occurring polynucleotide may be administered before, during, or after the onset of a disease or condition, and the timing of administration of the composition containing the therapeutic agent may vary. In some cases, the composition may be used as a prophylactic and may be administered continuously to a subject (e.g., a subject for immunization or a subject for treatment) who has a susceptibility to coronavirus or a tendency to a coronavirus-related condition or disease. Prophylactic administration may reduce the likelihood of the onset of an infection, disease, or condition, or may reduce the severity of the infection, disease, or condition.

[0091]

[0101] The non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition may be administered to a subject prior to the onset of symptoms. The non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition may be administered to a subject (e.g., a subject for immunization or a subject for treatment) after (e.g., as soon as possible thereafter) a test result, e.g., a test result resulting in a diagnosis, a test showing the presence of coronavirus in a subject (e.g., a subject for immunization or a subject for treatment), or a test showing the progression of a condition, e.g., a test showing decreased blood oxygen levels. The therapeutic agent may be administered after (e.g., as soon as possible thereafter) the onset of a disease or condition is detected or suspected. The therapeutic agent may be administered after (e.g., as soon as possible thereafter) a potential exposure to coronavirus, e.g., after a subject (e.g., a subject for immunization or a subject for treatment) has come into contact with an infected subject or knows that he or she has come into contact with an infected subject that may be contagious.

[0092]

[0102] The actual dosage level of the agent (e.g., a non-naturally occurring polynucleotide or pharmaceutical composition) of the present disclosure may be varied to obtain an amount of agent to achieve the desired therapeutic response for a particular subject, composition, and administration mechanism without toxicity to the subject (e.g., a subject for immunization or a subject for treatment). The selected dosage level may depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0093]

[0103] The dosage regimen may be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as dictated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suitable as a unit dosage for a subject (e.g., a subject for immunization or a subject for treatment); each unit contains a predetermined content of active agent calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form of the present disclosure may be determined by and directly depend on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the technical field of compounding such active agents for the treatment of susceptibility in an individual. The dosage may be determined by reference to the plasma concentration or local concentration of the cyclic polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined by reference to plasma or local concentrations of the linear polyribonucleotide or the antibody or antigen-binding fragment thereof.

[0094]

[0104] The non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition described herein may be in a unit dosage form suitable for single administration of a precise dosage. In the unit dosage form, the formulation may be divided into unit doses containing an appropriate content of the composition. In the unit dosage form, the formulation may be divided into unit doses containing an appropriate content of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage may be in the form of a package containing separate contents of the formulation. Non-limiting examples are packaged injection solutions, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. The formulations for injection disclosed herein may be in unit dosage form, for example, in ampoules, or in multi-dose containers with preservatives.

[0095]

[0105] In some cases, the increased level of a biologic in a subject is at least a 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold increase as determined by a diagnostic assay.

[0096]

[0106] Suitable diagnostic assays can include ophthalmic diagnostic assays, such as ophthalmic examinations, e.g., refraction, eye scans, ophthalmic coherence tomography, Farnworth-Munsell 100 Hue Test, computerized optic disc imaging and nerve fiber layer analysis (GDX, HRT, OCT), corneal topography, electroretinography (ERG), electronystagmography (EOG), visual evoked potentials (VEP), visual evoked responses (VER), fluorescein angiography, ophthalmic coherence tomography (OCT), retinal photography, fundus photography, specular microscopy, Goldmann, Humphrey, FDT, Octopus, biometry / IOL calculation, A-scan, B-scan, and combinations thereof.

[0097]

[0107] In some cases, retinal examination can be utilized.Non-limiting methods for evaluating retinal function and its changes include evaluating visual acuity (e.g., best-corrected visual acuity [BCVA], gait movement, navigation, object detection and identification), evaluating visual field (e.g., static and kinetic perimetry), performing clinical tests (e.g., slit lamp examination of anterior and posterior eye areas), evaluating electrophysiological response to all wavelengths of light and dark (e.g., all forms of electroretinography (ERG) [full field, multifocal and pattern], all forms of visual evoked potential (VEP), electronystagmography (EOG), color vision, dark adaptation and / or contrast sensitivity). Non-limiting methods for assessing anatomy and retinal health and changes therein include optical coherence tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measuring ocular motility and eye movements (e.g., nystagmus, fixation preference and stability), measuring reported outcomes (patient-reported changes in visually and non-visually guided behaviors and activities, patient-reported outcomes [PROs], quality of life, questionnaire-based assessments of daily activities, and measures of neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0098]

[0108] In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition reduces neoangiogenic signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to a comparable cell that has not been contacted with the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition. In some embodiments, the non-naturally occurring polynucleotide, AAV vector comprising the non-naturally occurring polynucleotide, AAV comprising the non-naturally occurring polynucleotide, or pharmaceutical composition reduces neoangiogenic signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to a comparable cell treated with a comparable VEGF inhibitor and a RTK / Tie2 or RTK / Tie2 activator encoded from two different non-naturally occurring polynucleotides. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition reduces vascular leakage in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to comparable cells that have not been contacted with the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition.In some embodiments, the non-naturally occurring polynucleotide, AAV vector comprising the non-naturally occurring polynucleotide, AAV comprising the non-naturally occurring polynucleotide, or pharmaceutical composition reduces vascular leakage in cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to comparable cells treated with a comparable VEGF inhibitor and a comparable RTK / Tie2 or RTK / Tie2 activator encoded from two different non-naturally occurring polynucleotides. In some embodiments, the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition reduces inflammation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to comparable cells that have not been contacted with the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition. In some embodiments, the non-naturally occurring polynucleotide, AAV vector comprising the non-naturally occurring polynucleotide, AAV comprising the non-naturally occurring polynucleotide, or pharmaceutical composition reduces inflammation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to comparable cells treated with a comparable VEGF inhibitor and a RTK / Tie2 or RTK / Tie2 activator encoded from two different non-naturally occurring polynucleotides.

[0099]

[0109] In some embodiments, the treatment methods described herein can treat ocular diseases. Relevant ocular diseases and conditions include, but are not limited to, blindness, color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Malacchia-Levenzenese (familial dominant drusen), and blue-cone monochromacy. In some embodiments, the ocular disease or condition is AMD. AMD can be wet AMD or dry AMD.

[0100]

[0110] In some cases, administration of the pharmaceutical composition is sufficient to reduce at least some symptoms of the disease or condition, treat the disease or condition, and / or eliminate the disease or condition. In some cases, the improvement of the disease or condition can be confirmed by any of the diagnostic assays provided. In other cases, the improvement can be obtained by interviewing the treated subject. For example, the subject may be able to tell the attending physician that his / her vision has improved compared to his / her vision before administration of the subject pharmaceutical. In other cases, an in vivo animal model can be used to confirm the reduction of the disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, dog models, and others. Pharmaceutical Compositions

[0111] Described herein is a pharmaceutical composition comprising the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, or the AAV comprising the non-naturally occurring polynucleotide described herein. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises two or more active agents disclosed herein. In some embodiments, the pharmaceutical composition comprising the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, or the AAV comprising the non-naturally occurring polynucleotide treats a disease or condition described herein. In some embodiments, the disease or condition comprises an eye disease. In some embodiments, the disease or condition comprises ocular ischemia syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiency, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Malacchia-Leventinese (familial dominant drusen), and blue-cone monochromacy.

[0101]

[0112] For in vivo delivery, the non-naturally occurring polynucleotide, AAV vector or AAV virion comprising the non-naturally occurring polynucleotide may be formulated into a pharmaceutical composition and generally administered intravitreally or parenterally (e.g., by routes of administration such as intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc.). In some embodiments, the pharmaceutical composition is formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymally, subcutaneously, intratumorally, pulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, rectally, orally, sublingually, transdermally, by inhalation, by inhalation spray form, by intracavitary-GI route, or a combination thereof.

[0102]

[0113] In some embodiments, the pharmaceutical composition can be used to treat a subject, such as a human or mammal, in need of treatment. In some cases, the subject can be diagnosed with a disease, such as an eye disease. In some embodiments, the subject pharmaceutical composition is co-administered with a second therapy. The second therapy can include any treatment for eye use. In some cases, the second therapy includes nutritional therapy, vitamins, laser treatment, such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, eyewear, eye drops, anesthetics, vision correction visual therapy, behavioral / perceptual visual therapy, and others. In some embodiments, any of the biologics previously described can be considered as a second therapy.

[0103]

[0114] In some embodiments, an effective amount of the pharmaceutical composition results in a reduction in the rate of loss of retinal function, anatomical integrity or retinal health, e.g., a 2-fold, 3-fold, 4-fold or 5-fold or greater reduction in the rate of loss and thus disease progression, e.g., a 10-fold or greater reduction in the rate of loss and thus disease progression.

[0104]

[0115] In some embodiments, an effective amount of the pharmaceutical composition reduces neovascularization signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neovascularization signaling in a cell that was not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces neovascularization in a subject in need of neovascularization treatment by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to neovascularization in the subject when the subject was not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces vascular leakage in a subject in need of treatment for vascular leakage by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the vascular leakage in the subject when the subject is not treated with the pharmaceutical composition. In some embodiments, an effective amount of the pharmaceutical composition reduces inflammation in a subject in need of treatment for inflammation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or more compared to the inflammation in the subject when the subject is not treated with the pharmaceutical composition.

[0105]

[0116] In some embodiments, an effective amount of a subject rAAV virion provides visual function, gain of retinal function, improvement in retinal anatomy or health, and / or improvement in ocular motility and / or improvement in neurological function, e.g., a 2-fold, 3-fold, 4-fold or 5-fold or greater improvement in retinal function, retinal anatomy or health, and / or improvement in ocular motility, e.g., a 10-fold or greater improvement in retinal function, retinal anatomy or health, and / or improvement in ocular motility. As will be readily appreciated by those of skill in the art, the dosage required to achieve the desired therapeutic effect is typically within the range of 1×10 8 ~Approx. 1×10 15 1×10 8 ~Approx. 1×10 15 This would be within the scope of recombinant virions.

[0106]

[0117] In some aspects, the compositions, e.g., pharmaceutical compositions, provided herein are administered to a subject in need thereof. In some cases, the administration is at least about 0.5×10 vectors. 9 vg, 1.0×10 9 vg, 1.0×10 10 , 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14 vg, 1.0×10 15 vg, 1.5×10 15 For example, for in vivo injection, e.g., direct injection into the eye, a therapeutically effective dose can be about 10 6 ~about 10 15 For example, approximately 10 AAV virions 8 ~10 12 For in vitro transduction, the effective amount of engineered AAV virions to be delivered to cells can be on the order of about 10 8 ~about 10 13Other effective dosages can be readily established by one of ordinary skill in the art by routine trials establishing dose-response curves.

[0107]

[0118] Administration can be repeated for any amount of time. In some embodiments, administration is twice daily, every other day, twice weekly, bimonthly, trimonthly, monthly, monthly, monthly, semiannually, annually, or biennially.

[0108]

[0119] Dosage treatment can be a single dose schedule or a multiple dose schedule.Furthermore, the subject can be administered an appropriate number of doses.The appropriate number of doses can be easily determined by those skilled in the art.In some embodiments, the pharmaceutical composition is administered by intravitreal injection, subretinal injection, microinjection or supraocular injection.

[0109]

[0120] In some embodiments, subjects may be screened for mutations by genetic testing before, during and / or after administration of the pharmaceutical compositions provided herein. Relevant genes that may be screened for mutations include RPE65, CRB1, AIPL1, CFH, or RPGRIP.

[0110]

[0121] In carrying out the treatment method or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease, disorder or condition to be treated, such as cancer.In some embodiments, the mammal is a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the efficacy of the therapeutic agent used, and other factors.The therapeutic agent and in some cases, the composition described herein can be used alone or in combination with one or more therapeutic agents, as a component of a mixture.

[0111]

[0122] The pharmaceutical compositions described herein can be administered to a subject by suitable routes of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal routes of administration.The compositions described herein can include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multiparticulate formulations and mixed immediate and controlled release formulations.

[0112]

[0123] The pharmaceutical compositions may be manufactured in a conventional manner, such as, by way of example only, using conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0113]

[0124] In certain embodiments, the pharmaceutical compositions provided herein include one or more preservatives for inhibiting microbial activity.Suitable preservatives include mercury-containing substances, such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0114]

[0125] In some embodiments, the pharmaceutical compositions described herein are formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In one aspect, the therapeutic agents described herein, e.g., therapeutic agents, are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives, such as preservatives, wetting agents, emulsifying agents, and dispensing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0115]

[0126] For intravenous injection or infusion or infusion, the pharmaceutical compositions described herein are formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hanks' solution, Ringer's solution or physiological saline buffer. For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0116]

[0127] Parenteral injection may involve bolus injection or continuous infusion. Pharmaceutical compositions for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers, with added preservatives. The compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous medium, and may contain formulatory agents, for example suspending agents, stabilizing agents and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable medium, for example sterile pyrogen-free water, before use.

[0117]

[0128] For administration by inhalation, the therapeutic agent is formulated for use as an aerosol, mist or powder. The pharmaceutical composition described herein is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer by using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, such as gelatin, for use in an inhaler or inhaler, can be formulated to contain the therapeutic agent described herein and a suitable powder base, for example, a powder mix of lactose or starch. The formulations containing the pharmaceutical composition are prepared as a solution in saline using benzyl alcohol or other suitable preservatives, fluorocarbons and / or other solubilizing or dispersing agents known in the art. Preferably, these compositions and formulations are prepared with suitable non-toxic pharmaceutically acceptable ingredients. The selection of suitable carrier depends on the exact nature of the desired nasal dosage form, for example, solution, suspension, ointment or gel.Nasal dosage form generally contains a large amount of water in addition to active ingredient.Small amounts of other ingredients, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffers and other stabilizers and solubilizers, may be present as necessary.Preferably, nasal dosage form should be isotonic with nasal secretions.

[0118]

[0129] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally adding suitable auxiliary if desired, grinding the resulting mixture, and processing the granulated mixture to obtain tablets or dragee cores.Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others, such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.If desired, disintegrants are added, such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or characterization of different combinations of active therapeutic agent doses.

[0119]

[0130] In another aspect, the dosage form comprises microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH modifiers, corrosion promoters, antifoaming agents, antioxidants, flavoring agents and carrier materials, such as binders, suspending agents, disintegrating agents, fillers, surface active agents, dissolving agents, stabilizers, lubricants, wetting agents and diluents.

[0120]

[0131] The liquid pharmaceutical dosage form for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharma-ceutically acceptable aqueous oral dispersion, emulsion, solution, elixir, gel and syrup.In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as (a) a disintegrant; (b) a dispersant; (c) a wetting agent; (d) at least one preservative, (e) a viscosity enhancer, (f) at least one sweetener and (g) at least one flavoring agent.In some embodiments, the aqueous dispersion further includes a crystal formation inhibitor.

[0121]

[0132] In some embodiments, the pharmaceutical compositions described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another phase, usually in the form of droplets. In general, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to excess water without any external mechanical dispersion or stirring. The advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Moreover, water or aqueous phase is added as needed just prior to administration, which ensures stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improved bioavailability of hydrophobic active ingredients.

[0122]

[0133] Buccal formulations are administered using various formulations known in the art.In addition, the buccal dosage forms described herein can further comprise a biodegradable (hydrolyzable) polymer carrier, which also functions to make the dosage form adhere to buccal mucosa.For buccal or sublingual administration, the composition can take the form of tablets, lozenges or gels that are formulated in a conventional manner.

[0123]

[0134] For intravenous injection, pharmaceutical compositions are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer, as necessary. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, suitable formulations preferably include aqueous or non-aqueous solutions with physiologically compatible buffers or excipients.

[0124]

[0135] Parenteral injection involves bolus injection or continuous infusion as required. Preparations for injection are presented in unit dosage form, for example in ampoules or multi-dose containers, with added preservatives as required. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous medium, and contain agents for formulation, for example suspending agents, stabilizing agents and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of the agent that modulates the activity of the carotid body in water-soluble form. Moreover, suspensions of the agent that modulates the activity of the carotid body are prepared as appropriate, for example as oily injection suspensions as required.

[0125]

[0136] Conventional formulation techniques include, for example, one or a combination of the following processes: (1) dry blending, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tabletting, extrusion, and others.

[0126]

[0137] In some embodiments, a pharmaceutical composition is provided that includes particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which upon mixing with water results in a substantially uniform suspension.

[0127]

[0138] In addition, the pharmaceutical composition may optionally contain one or more pH adjusting or buffering agents, including acids, such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers, such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amounts required to maintain the pH of the composition within an acceptable range.

[0128]

[0139] Additionally, the pharmaceutical compositions optionally contain one or more salts in an amount required to bring the osmolality of the composition into an acceptable range. Such salts include salts having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. kit

[0140] In some embodiments, the present specification discloses a kit for using the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition described herein.In some embodiments, the kit disclosed herein can be used to treat disease or condition in a subject.In some embodiments, the kit comprises a collection of materials or components other than the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, the AAV comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition.

[0129]

[0141] In some embodiments, the kits described herein include components for selecting a homogenous population of AAVs containing the non-naturally occurring polynucleotides described herein. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., AAV) synthesized and / or released by or expressed on the surface of a host cell. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition (e.g., cancer) disclosed herein in a subject. In some embodiments, the kits are configured specifically for the purpose of treating a mammalian subject. In some embodiments, the kits are configured specifically for the purpose of treating a human subject.

[0130]

[0142] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, or the pharmaceutical composition to a subject in need of administration. In some embodiments, the kit includes instructions for further engineering the cells to express a biological molecule (e.g., a non-naturally occurring polynucleotide, an AAV vector comprising a non-naturally occurring polynucleotide, or an AAV comprising a non-naturally occurring polynucleotide). In some embodiments, the kit includes instructions for thawing or otherwise restoring the biological activity of the non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, or the AAV comprising the non-naturally occurring polynucleotide, which may be cryopreserved, lyophilized, or cryo-hibernated during storage or transportation. In some embodiments, the kit includes instructions for measuring the viability of the restored non-naturally occurring polynucleotide, the AAV vector comprising the non-naturally occurring polynucleotide, or the AAV comprising the non-naturally occurring polynucleotide to ensure its effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0131]

[0143] If necessary, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings or other useful tools.The materials or components assembled in the kit can be provided to the practitioner in any convenient and suitable stored state that preserves their operability and usefulness.For example, the components can be in dissolved, dehydrated or lyophilized form; they can be provided at room temperature, refrigerated temperature or frozen temperature.The components are typically contained in suitable packaging material(s).

[0132]

[0144] The use of absolute or sequential terms, such as "shall," "will not," "shall," "shall not," "must," "must not," "first," "initial," "next," "thereafter," "before," "after," "finally," and "finally" are not intended to limit the scope of the embodiments disclosed herein, but are intended to be exemplary.

[0133]

[0145] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0134]

[0146] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0135]

[0147] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context can dictate a particular meaning.

[0136]

[0148] Any of the systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0137]

[0149] The term "about," when referring to a number or numerical range, means that the referenced number or numerical range is an approximation within experimental variability (or within statistical experimental error), and that the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range. In examples, the term "about" refers to ±10% of the stated number or value.

[0138]

[0150] The terms "increased," "increasing," or "increase" are used herein to generally mean an increase by a statically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase up to and including 100%, or any increase between 10-100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0139]

[0151] The terms "reduced", "reducing" or "reduction" are used herein to generally mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reduction" means a reduction of at least 10% compared to a reference level, e.g., a reduction of at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% compared to a reference level, or a reduction of up to and including 100% (e.g., non-existent or undetectable levels compared to a reference level), or any reduction between 10-100%. In the context of a marker or symptom, these terms contemplate a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably down to a level accepted as within the normal range for an individual without a given disease.

[0140]

[0152] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and explanation of the embodiments herein are not intended to be construed in a limiting sense. Thus, those skilled in the art will recognize numerous variations, changes, and substitutions without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It is understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is therefore contemplated that the present invention covers any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of such claims and their equivalents are thereby covered. EXAMPLES

[0141]

[0153] The following illustrative examples are representative of embodiments of the stimulation, systems and methods described herein and are not intended to be limiting in any way.

[0142] Example 1 AAV vector design and expression studies

[0154] Example 1 describes experiments to measure expression levels of a VEGF inhibitor in combination with either Ang1 protein (full length or fragment); or Ang2 inhibitory RNA (e.g., Ang2 shRNA), where Ang2 inhibitory RNA inhibition is determined by endogenous Ang2 expression levels.

[0143] Materials and Methods

[0155] Standard methods were used for molecular cloning of DNA constructs encoding VEGF antagonists (aflibercept, lucentis, anti-VEGF F(ab)' and single chain fragments (scFv) of variable regions, hCOMP-Ang1-FLD and hCOMP-Ang1-FLD-FLAG, Ang2 antibody scFv and Ang2 small hairpin RNA fragments). These proteins of interest (POIs) are listed in Table 1. Non-limiting exemplary AAV vectors containing different combinations of VEGF inhibitors and RTK / Tie2 activators for modulating Ang1 or Ang2 expression are listed in Table 2. Table 3 lists DNA primers used for PCR amplification and DNA sequencing analysis of the AAV vectors and expression cassettes described herein.

[0144] [Table 8-1]

[0145] [Table 8-2]

[0146]

Table 8-3

[0147]

Table 8-4

[0148]

Table 9-1

[0149]

Table 9-2

[0150]

Table 9-3

[0151]

Table 10-1

[0152]

Table 10-2

[0153]

Table 10-3

[0154]

Table 10-4

[0155]

Table 10-5

[0156]

Table 10-6

[0157]

Table 11-1

[0158]

Table 11-2

[0159]

Table 11-3

[0160]

Table 11-4

[0161]

Table 11-5

[0162]

Table 11-6

[0163]

Table 11-7

[0164]

Table 11-8

[0165]

Table 11-9

[0166]

Table 11-10

[0167]

Table 11-11

[0168]

Table 11-12

[0169]

Table 11-13

[0170]

Table 11-14

[0171]

Table 11-15

[0172]

Table 11-16

[0173]

Table 11-17

[0174]

Table 11-18

[0175]

Table 11-19

[0176]

Table 11-20

[0177]

Table 11-21

[0178]

Table 11-22

[0179]

Table 11-23

[0180]

Table 11-24

[0181]

Table 11-25

[0182]

Table 11-26

[0183]

Table 11-27

[0184]

Table 11-28

[0185]

Table 11-29

[0186]

Table 11-30

[0187]

Table 11-31

[0188]

Table 11-32

[0189]

Table 11-33

[0190]

Table 11-34

[0191]

Table 11-35

[0192]

Table 11-36

[0193]

Table 11-37

[0194]

Table 11-38

[0195]

Table 11-39

[0196]

Table 11-40

[0197]

Table 11-41

[0198]

Table 11-42

[0199]

Table 11-43

[0200]

Table 11-44

[0201]

Table 11-45

[0202]

Table 11-46

[0203]

Table 11-47

[0204]

Table 11-48

[0205]

Table 11-49

[0206]

Table 11-50

[0207]

Table 11-51

[0208]

Table 11-52

[0209]

Table 11-53

[0210]

Table 11-54

[0211]

Table 11-55

[0212]

Table 11-56

[0213]

Table 11-57

[0214]

Table 11-58

[0215]

Table 11-59

[0216]

Table 11-60

[0217]

Table 11-61

[0218]

Table 11-62

[0219]

Table 11-63

[0220]

Table 11-64

[0221]

Table 11-65

[0222]

Table 11-66

[0223]

Table 11-67

[0224]

Table 11-68

[0225]

Table 11-69

[0226]

Table 11-70

[0227]

Table 11-71

[0228]

Table 11-72

[0229]

Table 11-73

[0230]

Table 11-74

[0231]

Table 11-75

[0232]

Table 11-76

[0233]

Table 11-77

[0234]

Table 11-78

[0235]

Table 11-79

[0236]

Table 11-80

[0237]

Table 11-81

[0238]

Table 11-82

[0239]

Table 11-83

[0240]

Table 11-84

[0241]

Table 11-85

[0242]

Table 11-86

[0243]

Table 11-87

[0244]

Table 11-88

[0245]

Table 11-89

[0246]

Table 11-90

[0247]

Table 11-91

[0248] [Table 11-92]

[0249] [Table 11-93]

[0250] [Table 11-94]

[0251] [Table 11-95]

[0252] Maintenance of HEK293 LTV cells

[0156] HEK293 LTV cell line was cultured in DMEM medium containing 100 units / mL penicillin and 100 μg / mL streptomycin (P / S) (Corning) and 10% FBS (ATCC). It usually doubled in 24 hours. For regular maintenance, cells were split 1:10 once a week.

[0253] Transient transfection of HEK293 LTV cells with plasmid DNA In a 6-well plate, cells were seeded at 1×10^6 cells / well in 2 mL of DMEM medium containing 100 units / mL penicillin and 100 μg / mL streptomycin (P / S) and 10% FBS, and cultured overnight. On the day of transfection, the old medium was removed and replaced with Opti-MEM medium. Transient transfection was performed by diluting 1 μg shRNA plasmid and 1 μg Ang2 plasmid in 100 μL Opti-MEM and 4 μL PEI (1 μg / μL) in 100 μL Opti-MEM, mixing both diluted solutions, incubating for 10 minutes, and then adding dropwise to the cells. Four days after transfection, 500 μL medium was collected for assay and replenished with 500 μL fresh Opti-MEM medium. After another 3 days of incubation, all medium was collected for assay.

[0254] Enzyme-linked immunosorbent assay (ELISA) ELISA assay was performed as follows: 96-well plates were coated with 50 μL / well of capture antibody diluted in coating buffer (3.7 g sodium bicarbonate, 0.64 g sodium carbonate in 1 L Milli-Q water, pH 9.6) at 5 μg / mL overnight at 4° C. with a sealed cover. The next day, the coating solution was discarded and the plate was tapped on a paper towel to remove excess solution. 300 μL / well of blocking solution (commercial casein blocking buffer in PBS + 0.1% Tween 20) was added, the plate was sealed and incubated at 37° C. for 2 hours. After incubation, the blocking buffer was discarded and excess buffer was removed by tapping the plate on a paper towel. The samples to be tested were diluted in coating buffer, 50 μL / well of diluted sample was added and incubated at 37° C. for 2 hours. After incubation, the solution was discarded and the plate was tapped on a paper towel to remove excess solution. After washing six times with 300 μL / well of wash buffer (1×PBS containing 0.1% Tween-20, expires 30 days after preparation), the plate was tapped on a paper towel to remove excess solution, 50 μL / well of detection antibody diluted 1:100 in coating buffer was added, and the plate was incubated at 37° C. for 2 hours. After incubation, the solution was discarded and the plate was tapped on a paper towel to remove excess solution. 50 μL / well of streptavidin-HRP diluted 1:5000 in blocking buffer was added, and the plate was incubated at 37° C. for 1 hour. After incubation, the solution was discarded and the plate was tapped on a paper towel to remove excess solution. The plate was washed six times with 300 μL / well of wash buffer, and excess solution was removed by tapping the plate on a paper towel. Color reaction solution TMB was added at 50 μL / well, and the reaction was carried out for 15-20 min (or for a shorter period if color development was saturated) at room temperature in the dark.The color reaction was stopped by adding 50 μL / well stop solution, and the OD at 450 nm was read with the OD at 600 nm as reference 15 min after addition of stop solution.

[0255] Generation of recombinant baculovirus for AAV production

[0159] Recombinant baculovirus (rBV) was generated using the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, pFB shuttle plasmids containing the target genes were each diluted to 1 ng / μL in TE buffer, and 2 ng of each DNA was mixed with 20 μL of Δcath-DH10Bac competent bacteria (Virovek, Hayward, CA) containing the bacmid DNA molecule with the cathepsin gene deleted, incubated on ice for 30 minutes, followed by heat shock at 42°C for 30 seconds. After incubation on ice for 2 minutes, the bacteria were cultured at 37°C for 4 hours to recover, and then plated on agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. After 48 hours of incubation at 37°C, two white colonies containing recombinant bacmid DNA were picked and miniprep bacmid DNA was purified under sterile conditions. Approximately 5 μg of each bacmid DNA and 10 μL of GeneJet reagent (SignaGen Laboratories, Fredrick, MD) were each diluted in 100 μL ESFAF medium (Expression Systems, Davis, CA) and then mixed together for about 30 minutes to form a transfection mixture. Sf9 cells were plated in 6-well plates at 1.5e+6 cells / well in 2 mL ESFAF medium for about 30 minutes at 28°C. After removing the old medium from the Sf9 cells, each transfection mixture was diluted in 800 μL ESFAF medium and then added to the Sf9 cells. After overnight incubation at 28°C, an additional 1 mL ESFAF medium was added to each well. After a total incubation time of 4 days, the medium containing the rBV was collected and amplified at a 1:200 ratio to generate sufficient content of rBV ready for use in the AAV production process.

[0256] AAV production and purification

[0160] rBV carrying AAV2 Rep and mutant capsid genes and target expression cassettes, respectively, were used to co-infect Sf9-V432AG cells for AAV production. Briefly, 10 moi of rBV-Cap-Rep and 5 moi of rBV-target cassette were used to co-infect Sf9 cell line at a density of approximately 5e+6 cells / mL with 50% fresh ESFAF medium at a shaking speed of 180 rpm at 28°C in a shaker incubator for 3 days. At the end of infection, cell pellets were collected by centrifugation at 3,000 rpm for 10 minutes. The cells were lysed in Sf9 lysis buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100 and 125 units / mL Benzonase by vigorous vortexing followed by shaking at 350 rpm for 1 hour at 37°C. At the end of the shaking, the salt concentration was increased to 500 mM by vortexing and the lysate was clarified by centrifugation at 8,000 rpm for 20 minutes at 4°C. The clarified lysate was transferred to an ultraclear centrifuge tube for a SW28 swinging bucket rotor containing 5 mL of 1.50 g / cc and 10 mL of 1.30 g / cc cesium chloride solution. After approximately 18 hours of centrifugation at 28,000 rpm at 15°C, the AAV band was collected using a syringe and transferred to an ultraclear centrifuge tube for a 70ti centrifuge rotor. The tubes were filled with 1.38g / cc cesium chloride solution and heat sealed. The AAV samples were subjected to a second round of ultracentrifugation at 65,000 rpm at 15°C for approximately 18 hours, and the AAV band was collected using a syringe. The purified AAV samples were buffer exchanged into PBS buffer containing 0.001% Pluronic F-68 and filter sterilized using a 0.22um syringe filter. The sterilized AAV samples were stored at 4°C for up to one month, and then transferred to -80°C for long-term storage. The AAV titers were determined by real-time PCR method using a QuantStudio 7 Flex real-time PCR system (Invitrogen).

[0257] HEK293 transduction with AAV and ELISA quantification HEK293 cells were seeded on 6-well plates at 1.0E+6 cells / well in 2mL EMEM containing 10% FBS and incubated at 37°C and 5% CO2. After 24 hours, AAV was added to each well at 100,000vg / cell (MOI) and placed in an incubator. 24 hours after transduction, the old medium was replaced with fresh complete medium. Four days after transduction, culture supernatants were collected and an in-house ELISA developed to quantify VEGF-Trap and COMP-Ang1 proteins was performed. All transductions were performed in duplicate. For these transient expression or culture for the production of proteins for AAV transduction, HEK293 cell culture was performed in ultra-low IgG serum or serum-free medium.

[0258] Protein purification by column chromatography

[0162] Cell culture fluid of HEK293 transfected with plasmid DNA encoding POI or transduced with the corresponding AAV was filtered through a 0.2 μm filter to remove particulates and loaded onto a column (1 mL size) of MabSelect prismA protein A column chromatography at a flow rate of 1.5-2.0 mL / min. The column was washed with wash buffer (20 mM Tris-HCl, pH 7.3, 150 mM NaCl, 5 mM EDTA), eluted with elution buffer (0.1 M sodium acetate, pH 3.0-3.6), and neutralized with 1 / 10 neutralization buffer (1.0 M Tris-HCl, pH 10). The neutralized protein solution was buffer exchanged into 1×PBS containing 0.01% (w / v) Pluronic F68 or Tween 20 and sterile filtered through a 0.2 μm syringe filter pre-wetted with PBS. The final preparation was stored at -80°C.

[0259] Results - Plasmids constructed for this study

[0163] A total of 16 plasmids were constructed for this project to study the function of aflibercept, angiopoietin 1 and 2 and their synergetic effects in neovascularization. The constructed plasmids include AMI071, AMI077, AMI136, AMI142, AMI143, AMI144, AMI145, AMI146, AMI147, AMI148, AMI149, AMI150, AMI151, AMI152, AMI153 and AMI154, AMI155, AMI156, AMI157, AMI158, AMI159, AMI160, AMI161, AMI162, AMI163, AMI166, AMI167 and AMI169. Detailed AAV construct sequences and their respective regulatory elements are listed in Table 4.

[0260] Optimized Ang1 coding sequence improved protein expression

[0164] The non-optimized and optimized hCOMP-Ang1 sequences were cloned into the same plasmid backbone to create AMI071 and AMI077. Recombinant baculovirus was generated and used to infect Sf9 cells to produce AAV. Purified AAV2.N54 vector was used to transduce HEK293 cells, and Ang1 protein levels were determined using ELISA assay. The results are shown in Table 5, which show that the optimized Ang1 coding sequence improved protein expression levels by more than 5-fold.

[0261] [Table 12]

[0262] Custom-designed shRNA against Ang2 inhibited Ang2 expression A series of shRNAs against human Ang2 gene were cloned under the control of human U6 promoter. Plasmids containing these shRNAs were transfected into HEK293 cells in 6-well plates together with a plasmid expressing human Ang2 protein. Four days after transfection, 500 μL of medium from each well was collected, the same volume of fresh medium was added to the well, and the cells were cultured for another 3 days. Then, all the medium was collected and the expression of VEGF-Trap and human Ang2 was determined by ELISA assay. The results of Ang2 shRNA are shown in Table 6. The results demonstrate that all shRNAs have an inhibitory effect on Ang2 expression, among which shRNA3 and shRNA4 are the most efficient. When all the plasmids were packaged into AAV2.N54 vector, the resulting vector was transduced into HEK293 cells for 4 days. All the shRNAs show an inhibitory effect on Ang2 expression. shRNA3 and shRNA4 show the most efficient inhibitory effect on Ang2 expression (Table 7).

[0263] [Table 13]

[0264] [Table 14]

[0265] The dual cassette performed better than the fusion protein construct for targeted gene expression To target multiple pathways, both VEGF-Trap and angiopoietin 1 (Ang1) genes were cloned into one plasmid flanked by both AAV ITRs in a dual cassette or fusion protein configuration. These plasmids were used to generate AAV2.N54 vectors. In the dual cassette configuration, VEGF-Trap and Ang1 were each driven by a CMV enhancer / promoter followed by an SV40 intron, respectively, and terminated by a synthetic polyA sequence. In the fusion protein configuration, VEGF-Trap protein was fused to either furin and F2A sequence or Ang1 by a 4-unit GGGGS linker. The former configuration produced two separate proteins after translation by cleavage at the F2A site (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 15). The latter configuration produced a single fusion protein after translation. The results indicate that the dual cassettes produced higher protein expression for VEGF-Trap (AMI136 and AMI153) than fusion protein constructs with either furin-F2A (AMI142 and AMI154) or 4xGGGGS linker (AMI144). The furin-F2A cleavage polypeptide sequence comprises the polypeptide sequence of RRKRKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 16). The dual construct with the optimized Ang1 coding sequence (AMI153) produced higher Ang1 expression than any other construct, but VEGF-Trap was reduced (Table 8 and Figure 5). Tables 9 and 10 summarize the additional AAVs and their VEGF inhibitory effects in combination with either increased Ang1 expression (Table 9); or reduced Ang2 by Ang2 shRNA (Table 10, Ang2 shRNA1-6).

[0266] [Table 15]

[0267] [Table 16]

[0268] [Table 17]

[0269] Example 2 Therapeutic efficacy of AAV vectors containing non-naturally occurring polynucleotides in a laser-induced choroidal neovascularization (CNV) model in mice Example 2 describes the assessment of inhibition of neovascularization in a laser-induced model of choroidal neovascularization (CNV) in a mouse (Mus musculus; C57BL / 6J; male; 8-12 weeks old) model.

[0270] treatment

[0168] Control: AAV2.N54.Δ120 carrying the null mutated AVMX-110 (the "sham" vector) will be used at an intermediate dose, 4e+8vg / eye.

[0271] Test Articles: AAV vectors carrying different transgenes will be evaluated at a concentration of 4e+8 vg / eye. Each vector will be diluted in formulation buffer at 4e+8 vg / μl.

[0272] Dosing: Mice will be dosed bilaterally with AAV 28 days prior to laser. Vehicle will be dosed 3 days prior to laser. AAV preparations will be drawn from vials through a 5 μm filter (B Braun filter needle (FN5120) 5 micron filter in a female Luer lock connector with a 20 Ga.×1 and ½ inch (in.) thin wall needle for withdrawal or injection of medication from rubber stoppered vials (product code: 415025), or equivalent filter needles are acceptable). Table 17 describes the CNV study experimental design. Table 18 summarizes the testing system including animals, housing and environmental conditions. Table 19 describes the food and water provided to mice used in the CNV study.

[0273] [Table 18]

[0274] [Table 19]

[0275] [Table 20]

[0276] Animal health and acclimation

[0171] Animals will be acclimated to the study environment for a minimum of three days. Upon completion of the acclimation period, each animal will be physically examined by a laboratory animal technician to determine suitability for study participation. Examinations will include, but are not limited to, skin and external ear, eyes, abdomen, behavior, and general condition. Animals determined to be in good health will be released into the study.

[0277] Randomization and study identification

[0172] Animals will be randomly assigned to study groups according to the institution's Standard Operating Procedures (SOPs). Animals will be uniquely identified by corresponding cage card numbers, ear punches and numbers.

[0278] Intravitreal injection On day -28 prior to injection, mice will be given buprenorphine 0.01-0.05 mg / kg SQ. Animals will then be sedated for intravitreal injection and one drop of 0.5% proparacaine HCL will be applied to both eyes. Alternatively, mice may be anesthetized with inhaled isoflurane. The conjunctiva will be gently grasped with Dumont #4 forceps and the injection will be made using a 33G needle and Hamilton syringe. After dispensing the syringe contents, the syringe needle will be slowly withdrawn. After the injection procedure, one drop of ofloxacin ocular solution will be applied topically to the surface of the eye along with eye lube.

[0279] Laser-Guided CNV Procedure On day 0, mice will receive buprenorphine 0.01-0.05 mg / kg SQ. Topical mydriatics (1.0% tropicamide HCL and 2.5% phenylephrine HCL) will be applied at least 15 minutes before the laser procedure. Mice will be sedated with an intraperitoneal injection of ketamine / xylazine. The cornea will be kept moist using topical eyewash, and body temperature will be maintained using a hot pad. A 532 nm diode laser delivered through a slit lamp will be used to create four single laser spots surrounding the optic nerve. Both mouse eyes will undergo laser treatment on day 0 according to the schedule in the experimental design. Eye lubricant will be placed after the laser.

[0280] Parameters to be measured Testing. Mortality and morbidity will be observed daily with cageside observations with special attention paid to both eyes.

[0281] Fluorescein angiography (FA). FA will be performed on both eyes on the 7th day after laser. Pupil dilation for FA will be performed using topical mydriatics (1.0% tropicamide HCL and 2.5% phenylephrine HCL; one drop in each eye 15 minutes before testing). Mice will be sedated with an intraperitoneal injection of ketamine / xylazine. Retinal photography will be performed approximately 1 minute after intravenous sodium fluorescein injection (12 mg / kg).

[0282] Euthanasia. At the time points in the experimental design table above, animals will be euthanized by carbon dioxide asphyxiation and death will be confirmed by cervical dislocation. After euthanasia, both eyes of selected animals will be collected for flat mount analysis or PK histology.

[0283] Ocular Tissue Collection for Homogenization. Eyes will be enucleated and retina and RPE / choroid areas will be dissected from fresh eyes and snap frozen. Tissues will be placed into appropriate pre-weighed and labeled analysis vials and placed on dry ice until immediately re-weighed to determine sample weight and transferred to the freezer. Samples will be weighed on a balance capable of measuring to 4 decimal places. Serum (2 mL polypropylene screw-cap tubes) and retina / RPE / choroid / sclera ("eye cup") (2 mL Precellys homogenization tubes) will be collected. Samples will be homogenized in phosphate buffered saline (PBS). A Precellys Evolution benchtop homogenizer will be used (3 x 6500 rpm [each cycle for 30 seconds], delay for 30 seconds) and samples will be returned to the -80°C freezer.

[0284] Example 3 In vitro and in vivo bioanalytical analysis of non-GLP AVMX-112 (Ang1) Example 3 describes studies for in vitro expression of AVMX-112 (a dual gene construct for expressing aflibercept and Ang1). In vitro permeability assays using FITC-dextran showed significant protection from leakage in the presence of Ang1. In vivo mouse laser-induced choroidal neovascularization (CNV) model showed significant wound healing with AVMX-112. Table 20 describes the constructs used in studies utilizing AVMX-112.

[0285] [Table 21]

[0286] The constructs used in this study expressed dual genes. The common genes of interest (GOI) were aflibercept (AVMX-110) and human Ang1. Ang1 full-length protein has a tendency to form aggregates; therefore, a shorter sequence has been used to produce Ang1 consisting of aa 284-498. The sequence retains part of the coiled-coil domain of rat cartilage oligomeric matrix protein (COMP) at its N-terminus. Figure 10A illustrates exemplary information regarding the disadvantages of Ang1, COMP-Ang1, and full-length Ang1. Figure 10B illustrates an exemplary dual expression AAV construct. Each GOI was flanked by two separate CMV promoters, so aflibercept and COMP-Ang1 were expressed as separate proteins. AMI071 and AMI077 were COMP-Ang1 constructs without aflibercept. These were used in this study for in vitro expression, not dual gene constructs. AVMX-110 expresses only aflibercept and was used for in vitro and in vivo efficacy comparisons.

[0287] Mechanism of action CNV is the pathological growth of new blood vessels from existing choroidal vessels. This results in vision loss in the later stages. Vascular endothelial growth factor (VEGF) plays a leading role in the pathological progression of CNV. Aflibercept (Eylea) protein is one of the leading protein drugs available for the treatment of CNV. However, aflibercept and other anti-(anit-)VEGF drugs have disadvantages such as the need for repeated and continuous administration, or refractory state / tachyphylaxis, which is a rapid diminishing response to successive doses. Therefore, there is a need for other mechanisms that can support aflibercept anti-VEGF activity. Angiopoietins, particularly Ang1, facilitate non-leaky, non-inflammatory, functional and stable blood vessels. Ang1 reduces inflammation-induced vascular leakage and inflammatory cell infiltration by tightening cell junctions and reducing adhesion molecules. Table 21 lists the materials used in the AVMX-112 study. All AAV constructs for this example were produced in Sf9 and subjected to two cycles of CsCl ultracentrifugation. The titers of the constructs were checked by using qPCR.

[0288] [Table 22]

[0289] In vitro expression and quantification HEK293 cells were transduced with the AAV constructs mentioned in Table 20. After transduction, supernatants were collected and aflibercept and COMP-Ang1 expression levels were quantified.

[0290] In vitro vascular permeability assay

[0183] An in vitro vascular permeability assay was performed using human umbilical vein endothelial cells (HUVEC). 5.0E+04 cells were plated onto the apical compartment of a 24-well transwell plate in serum-free medium. The transwell apical compartment was pre-coated with collagen before plating the cells according to a standard protocol (Application Note 26 "Fabrication of Collagen I Gels," ibidi USA, Inc., Fitchburg, WI). After 72 hours of incubation in a 37°C incubator, the cells were treated with 20 ng / mL VEGF in the presence and absence of 253 ng / mL (5-fold molar concentration of VEGF) aflibercept and / or 250 ng / mL (5-fold molar concentration of VEGF) COMP-Ang1 protein for 1 hour. After treatment, the medium was replaced with fresh medium in the basolateral chamber and 1 mg / mL FITC-dextran was added to the apical chamber and incubated for 30 min. After 30 min, 50 μL was taken from the basolateral compartment and supplemented with 300 μL of phenol red-free DMEM. 100 μL of this sample was transferred to a black 96-well plate. Fluorescence intensity at 490 / 520 nm excitation / emission spectrum was measured in triplicate readings.

[0291] In vivo mouse CNV model For in vivo studies, the mouse CNV model was used as described previously, and the different constructs were injected intravitreosly (IVT) at a dose of 4E+08vg / eye. The AAV constructs were injected 28 days before FA analysis of the ANG1 construct and comparison of its expression with AVMX-110 and aflibercept in serum and eye samples were obtained from animals that were intravitreally (IVT) injected with non-GLP AAV constructs carrying different gene products for efficacy studies in the mouse CNV model. The titer of the IVT injection was 4.8E+08vg / eye in all groups. The groups involved in which the study was performed are shown in Table 22.

[0292] [Table 23]

[0293] Homogenization of eye samples Ocular and serum samples were obtained after euthanasia. The homogenized tissue was further sonicated by keeping the samples on ice and sonicating three times for 20 seconds with 20 second intervals. The sonicated ocular samples were then centrifuged at 13,000 rpm for 3-4 minutes. The supernatant was then collected and used for the determination of aflibercept levels using the standardized VEGF-Trap ELISA mentioned in the introduction section.

[0294] In vitro expression in HEK293 cells Aflibercept and Ang1 expression in double gene constructs were compared with single gene constructs (Figure 11). Table 23 describes the expression profile of aflibercept and Ang1 in all constructs used in the study. Non-optimized ANG1 construct (AMI136) produced higher aflibercept but lower Ang1, while AMI153 had equal expression of aflibercept and ANG1. Statistical analysis was performed using GraphPad Prism software.

[0295] [Table 24]

[0296] In vitro vascular permeability assay

[0187] In vitro permeability assay showed the effect of different proteins on the permeability of FITC-dextran through HUVEC cell monolayer. Instead of AAV construct, purified protein was used in the assay. Figure 12 illustrates that VEGF promoted leakage, but aflibercept and Ang1 acted to reduce the leakage of FITC-dextran. Ang1 had significantly higher leakage protection compared to aflibercept alone. In summary, aflibercept and Aed1 acted to significantly reduce permeability compared to VEGF or VEGF combined with aflibercept.

[0297] Fluorescein angiography (FA) analysis FA data after laser injury were compared between groups. Statistical analysis was performed by comparing all other groups with vehicle control. Figure 13A and Figure 13B show Ang1 and VEGF-Trap construct comparison as bar graphs ± SEM, and statistical analysis using one-way ANOVA, and results of multiple comparisons using Dunnett's test. Figure 14 illustrates representative FA images from different groups. When vehicle was used to compare the rest of the groups, there was no significant difference between the vehicle and sham control groups, as expected. However, animals in the AAV2.N54-120 group showed significant laser injury recovery. When AAV2.N54-120 group animals were excluded from the analysis, AAV2.N54-153 animals also showed significant differences with vehicle group animals. Lesion area and p-values ​​for different groups are summarized in Table 24.

[0298] [Table 25]

[0299] VEGF-Trap levels in ocular samples

[0189] VEGF-Trap concentration was expressed as pg of aflibercept per eye cup. The eye cup consisted of retina, sclera, choroid and retina (Figure 15). Expression in AVMX-110, AAV2.N54-aflibercept, showed higher levels of aflibercept expression compared to other groups (Table 25).

[0300] [Table 26]

[0301] In conclusion, in vitro expression showed a significant increase in the expression of Ang1 after codon optimization, whether it was a single or double gene construct. In vitro permeability assay also showed significant leakage protection by Ang1. Aflibercept and Ang1 worked synergistically to reduce leakage caused by VEGF. AVMX-110 efficiently reduced the lesion area in the mouse CNV model. The Ang1 construct AMI153 also showed efficacy comparable to AVMX-110, even though the aflibercept expression of AMI153 was much lower than that of AVMX-110. However, AMI136, which had a higher aflibercept expression compared to AMI153, did not show efficacy. This explains the importance of Ang1 in this model.

[0302]

[0191] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to one skilled in the art upon reading this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, any of the techniques and devices described above can be used in various combinations. Any publications, patents, patent applications and / or other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application and / or other document was individually and separately indicated to be incorporated herein by reference for all purposes.

Claims

1. a) a VEGF inhibitor, and b) A non-naturally occurring polynucleotide comprising one or more expression cassettes for expressing receptor tyrosine kinase (RTK) / Tie2 or an activator of RTK / Tie2.

2. The non-naturally occurring polynucleotide of claim 1, wherein a) and b) are expressed as separate polypeptides comprising a VEGF inhibitor and an RTK / Tie2 or RTK / Tie2 activator, or as contiguous polypeptides that can be cleaved into separate polypeptides.

3. 3. The non-naturally occurring polynucleotide of claim 2, wherein the contiguous polypeptide comprises a protease-cleavable sequence, a furin-cleavable sequence, or a self-cleaving polypeptide sequence.

4. 2. The non-naturally occurring polynucleotide of claim 1, wherein the VEGF inhibitor comprises an anti-VEGF antibody or VEGF-TRAP.

5. 2. The non-naturally occurring polynucleotide of claim 1, wherein the activator of RTK / Tie2 comprises angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), angiopoietin-3 (Ang-3), angiopoietin-4 (Ang-4), or a functional fragment of any one of them.

6. 6. The non-naturally occurring polynucleotide of claim 5, wherein Ang-1 comprises or consists of a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

3.

7. 6. The non-naturally occurring polynucleotide of claim 5, wherein the functional fragment of Ang-1 comprises or consists of a fibronectin-like domain (FLD).

8. 8. The non-naturally occurring polynucleotide of claim 7, wherein the FLD comprises or consists of a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

5.

9. 23. The non-naturally occurring polynucleotide of claim 21 or 22, wherein the FLD is fused to a soluble polypeptide.

10. 10. The non-naturally occurring polynucleotide of claim 9, wherein the soluble polypeptide comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:2 or SEQ ID NO:

1.

11. 2. The non-naturally occurring polynucleotide of claim 1, wherein the activator of RTK / Tie2 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:

6.

12. 13. The non-naturally occurring polynucleotide of claim 12, wherein the activator of RTK / Tie2 comprises an antibody or a fragment thereof.

13. 13. The non-naturally occurring polynucleotide of claim 12, wherein the activator of RTK / Tie2 binds to and inhibits Ang-2.

14. 13. The non-naturally occurring polynucleotide of any one of claims 12, wherein the antibody or fragment thereof comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 139-141, a fragment thereof, or a combination thereof.

15. 2. The non-naturally occurring polynucleotide of claim 1, wherein the activator of RTK / Tie2 comprises an inhibitory RNA.

16. 16. The non-naturally occurring polynucleotide of claim 15, wherein the inhibitory RNA comprises an shRNA, an siRNA, an miRNA, or a combination thereof.

17. 2. The non-naturally occurring polynucleotide of claim 1, wherein the one or more expression cassettes comprise one or more promoters, one or more internal ribosome entry sites (IRES), or both.

18. A viral vector comprising the non-naturally occurring polynucleotide of any one of claims 1 to 17.

19. The viral vector of claim 18, which is an adeno-associated viral (AAV) vector.

20. 20. Use of the non-naturally occurring polynucleotide of any one of claims 1 to 17, or the viral vector of claim 18 or 19, in the manufacture of a medicament for use in a method for treating a disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the non-naturally occurring polynucleotide or the viral vector to a subject. thereby treating diseases or conditions including ocular ischemia syndrome, proliferative retinopathy, neovascular glaucoma (NG), uveitis, neovascular uveitis, color vision deficiency, age-related macular degeneration (nAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), retinal vein occlusion (RVO), glaucoma, Bardet-Biedl syndrome, Best's disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum's disease, Stargardt's disease, Usher's syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi's disease, Malaccia-Levensteine ​​(familial dominant drusen), or blue-cone monochromatic color deficiency.

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